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Pathogenetische Untersuchungen zur Ausbildung unterschiedlicher Phänotypen und zur Vermehrung humaner Mastzellen bei Wundheilung und UrtikariaHermes, Barbara 04 December 2001 (has links)
Bei der Wundheilung und fibrosierenden Prozessen sowie bei der Urtikaria ist eine Mastzellvermehrung bekannt. Mastzellen (MZ) üben bei der Urtikaria eine Schlüsselfunktion aus und scheinen auch zum Bindegewebsumbau beizutragen. In humanem Narbengewebe (5-369 Tage alt) wurden MZ-Zahlen und MZ-Subpopulationen mittels Enzym- und Immunhistochemie im Vergleich zu normaler Haut untersucht. Außerdem wurden in Gewebsextrakten Aktivität und mRNA-Expression der MZ-Proteasen und in vitro ihre mitogene Wirkung auf Fibroblasten und Keratinozyten bestimmt. Zur Klärung von Mechanismen, die zur MZ-Vermehrung beitragen könnten, analysierten wir die Expression von MZ-Chemoattraktoren und MZ-Wachstumsfaktoren sowie ihrer Rezeptoren in humanem Narbengewebe (a), läsionaler und nicht-läsionaler Urtikariahaut (b) und in normaler Haut (c): SCF, c-Kit, NGF-R TrkA, NGF-R p75, GM-CSF, GM-CSF-R (a, b, c); NGF, TGF-(, TGF-(-R I, TGF-(-R II (a,c) mittels Immunhistochemie (a, b, c) und RT-PCR (a, c). Zusätzlich wurde die Expression der proentzündlichen Zytokine IL-3, -8, TNF-( untersucht (b, c). Tryptase und Chymase enthaltende MZ waren in Narben gegenüber normaler Haut signifikant vermindert ebenso wie Chymaseaktivität und -mRNA-Expression in Narbengewebsextrakten. Die Anzahl Tryptase-haltiger MZ war unverändert, obwohl Tryptaseaktivität und -mRNA in Narben vermehrt waren. Beide Proteasen erhöhten in vitro die mitogene Antwort von Fibroblasten, jedoch nicht von Keratinozyten. c-Kit+-MZ fanden sich in der mittleren und tiefen Dermis von Narben signifikant vermehrt. SCF, TGF-(, TGF-(-R I und II, NGF-R p75 und TrkA zeigten sich sowohl immunhistochemisch als auch in der RT-PCR in Narbengewebe hochreguliert im Vergleich zu normaler Haut, wohingegen NGF, GM-CSF und GM-CSF-R nur schwach exprimiert waren ohne Unterschied zwischen beiden Geweben. Mittels FACS-Analyse wurde erstmalig die Expression von TGF-(-R I und II auf isolierten Haut-MZ nachgewiesen. Im Gegensatz zu diesen Befunden waren in Urtikariagewebe SCF- und NGF-R p75-exprimierende Zellen vermindert im Vergleich zu normaler Haut. Die Zahl von c-Kit+-, NGF-R TrkA+-, GM-CSF+- und GM-CSF-R+ -Zellen zeigte sich unverändert. Hingegen war die Expression von IL-3 und TNF-( auf Endothelzellen in läsionaler und nicht-läsionaler Urtikariahaut signifikant hochreguliert. Die dargestellten Ergebnisse mit signifikanter Verminderung von Chymase- und Tryptase-haltigen MZ in humanem kutanen Narbengewebe sprechen für MZ-Degranulation nach Trauma. Nachfolgend findet sich in Narbengewebe eine Chymase--, Avidin--, Tryptase+-, c-Kit+-MZ-Subpopulation, am ehesten Folge einer Einwanderung und Proliferation von unreifen MZ oder MZ-Vorläufern, die von den vermehrt exprimierten Wachstumsfaktoren SCF und TGF-(, eventuell auch von NGF über seine vermehrt exprimierten Rezeptoren, induziert werden könnten. Neben NGF und TGF-( scheint auch SCF eine Rolle bei der Wundheilung zu spielen. Bei entzündlichen Hautkrankheiten unterschiedlicher Prägung wie Wundheilung und Urtikaria liegen offenbar verschiedenartige Regulationsmuster der MZ-Proliferation und -Differenzierung vor. Unsere Ergebnisse legen nahe, dass bei Trauma Feedbackmechanismen über Wachstumsfaktoren wie SCF, TGF-( und NGF und ihre Rezeptoren auf MZ ablaufen, bei der Urtikaria unter Mitberücksichtigung bereits bekannter Daten aus der Literatur vorzugsweise über eine Interaktion von Mast- und Endothelzellen. / In wound healing and fibrosing processes as well as in urticaria an increase of mast cells (MC) has been observed. MC are key-players in urticaria, and might also contribute to tissue repair. In human cutaneous scar tissue (5-369 days old) and normal skin MC dynamics and MC subtypes were analysed by enzyme- and immunohistochemistry. Moreover, the activity of the MC proteases in extracts of both tissues and their in vitro effect on the mitogenesis of fibroblasts and keratinocytes were assessed. To elucidate mechanisms involved in mast cell accumulation, expression of MC chemotaxins, MC growth factors and their receptors was evaluated comparing cutaneous scar tissue (a), lesional and non-lesional skin of urticaria (b) and normal skin (c): SCF, c-Kit, NGF-R TrkA, NGF-R p75, GM-CSF, GM-CSF-R (a, b, c); NGF, TGF-(, TGF-(-R I, TGF-(-R II (a,c) by immunohistochemistry (a, b, c) and by RT-PCR (a, c). Additionally, expression of proinflammatory cytokines (IL-3, -8, TNF-() was studied (b, c). Tryptase and chymase containing MC were markedly decreased in scars as well as chymase activity and mRNA expression, whereas overall numbers of tryptase containing MC did not differ from those in normal skin, although tryptase activity and mRNA expression were increased in scar extracts. Both proteases induced a dose-dependent mitogenic response in 3T3-fibroblasts, but not in HaCaT-keratinocytes. Numbers of c-Kit+ MC were significantly increased in the mid and lower dermis of scars. Furthermore, SCF, TGF-(, its receptors I and II, the NGF-R p75 and TrkA were shown to be upregulated in scars both by immunohistochemistry and by RT-PCR, while NGF, GM-CSF and the GM-CSF-R were only weakly expressed without differences between scar and normal tissue. In addition, expression of TGF-(-R I and II could be shown on isolated human skin MC by FACS-analysis. In contrast to these findings, SCF- and NGF-R p75-expressing cells in urticaria tissue were downregulated compared to normal skin. Numbers of c-Kit+, NGF-R TrkA+, GM-CSF+ and GM-CSF-R+ cells remained unchanged. However, IL-3 and TNF-( expression was upregulated on endothelial cells in lesional and non-lesional skin of urticaria. These data show that numbers of resident MCTC are very low in human cutaneous scars suggesting massive mediator release from these cells after wounding. Instead, scar tissue is populated by a chymase-, avidin-, tryptase+, c-Kit+ MC subpopulation that is reflecting most probably an immigration and / or proliferation of immature MC and their precursors which might be promoted by SCF and TGF-beta, possibly also NGF via its receptors. Next to TGF-( and NGF, also SCF seems to play a role in wound healing. Our findings suggest different regulation patterns of MC increase in inflammatory conditions of the skin. After wounding, feedback mechanisms via growth factors (SCF, TGF-(, possibly NGF) and their receptors on MC could be operative, while in urticaria in accordance with data from the literature interactions between MC and endothelial cells appear to be essential.
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Dissection of TGF-beta/Smads in the renal inflammation and fibrosis. / 转化生长因子/Smads信号蛋白在肾脏炎症和纤维化中的作用 / CUHK electronic theses & dissertations collection / Zhuan hua sheng zhang yin zi/Smads xin hao dan bai zai shen zang yan zheng he xian wei hua zhong de zuo yongJanuary 2012 (has links)
目的: 转化生长因子-1(TGF-β1)通过与II型受体结合而引起I型受体活化,进一步激活其下游信号分子蛋白Smad2 和Smad3,它们与Smad4(Co-Smad)结合后形成Smad复合体并发生核转移,从而发挥广泛的生物学效应。同时,整个TGF-β信号通路又受到其抑制因子Smad7的负反馈调节。研究结果显示Smad3是肾脏炎症和纤维化中重要的致病分子,相反,Smad7在多种肾脏疾病中起保护作用。然而,由于转化生长因子II型受体(TβRII),Smad2 或Smad4基因敲除的小鼠无法存活,这些分子在TGF-β1介导的肾脏炎症和纤维化中的功能尚未见报道。因此,本研究旨在剖析TβRII、Smad2 和Smad4 在肾脏疾病发生发展中的作用及机制。 / 方法:本研究利用Cre/LoxP系统分别靶向敲除小鼠肾小管上皮细胞的TβRII、Smad2 或者Smad4,通过结扎小鼠单侧输尿管建立梗阻性肾病模型,观察这些分子对肾脏炎症和纤维化的影响,并用体外实验进行验证。具体实验结果请参见本论文第III,IV, V章。 / 结果:通过分析,本论文取得以下新的发现: / (1) TβRII在TGF-β1介导的肾脏炎症和纤维化的双向调节中起到了决定性的作用:研究结果显示条件性敲除TβRII明显抑制TGF-β/Smad3介导的肾脏纤维化,同时增强NF-κB引起的肾脏炎症反应。由此可见,TRII不仅仅是TGF-β/Smad信号通路的启动因子,更决定了TGF-β1对肾脏炎症和纤维化的双向性调节。(参见第III章) / (2)尽管Smad2和Smad3结构相似并共同介导了TGF-β1的生物学效应,本研究意外发现Smad2可反向调节Smad3引起的纤维化。体内和体外实验共同证实,敲除Smad2基因增强了Smad3的磷酸化,核转位及其转录子活性,并能促进Smad3与I型胶原转录子的结合,进而加重肾脏纤维化(参见第IV章)。 / (3)我们还发现Smad4不仅作为TGF-β/Smad信号通路的共有蛋白,它在TGF-β1介导肾脏炎症和纤维化中起到了重要的双向性调节作用:条件敲除Smad4显著降低了Smad7对NF-κB介导肾脏炎症的抑制作用,同时在转录水平(而非磷酸化水平)抑制Smad3的功能,从而减轻纤维化。(参见第V章) / 结论:TβRII和Smad4 在TGF-β1介导肾脏炎症和纤维化中起到了重要的双向性作用;Smad2通过抑制Smad3信号传导和功能,在肾脏纤维化中起保护作用。 / Objectives: TGF-β1 binds its receptor II (TβRII) and then activates receptor I to initiate the downstream Smad signaling, called Smad2 and Smad3 which bind a common Smad4 to form the Smad complex and then translocate to nucleus to exert its biological activities. This process is negatively regulated by an inhibitory Smad7. While the pathogenic role of Smad3 and the protective role of Smad7 in renal fibrosis and inflammation are clearly understood, the functional role of TβRII, Smad2 and Smad4 in kidney diseases remains largely unexplored due to the lethality of these knockout mice. Therefore, the aim of present study is to dissect the functional role of these TGF-β/Smad signaling molecules in renal inflammation and fibrosis. / Methods: Kidney conditional knockout (KO) mice for TβRII, Smad2 and Smad4 were generated by crossing the FloxFlox mice with the kidney specific promoter driven Cre (KspCre) mice, in which TβRII, Smad2 or Smad4 were specifically deleted from the kidney tubular epithelial cells (TEC) respectively. Then, a well-characterized progressive renal inflammation and fibrosis mouse model of Unilateral ureteral obstructive (UUO) nephropathy was induced in these conditional KO mice and the specific roles for TβRII, Smad2, and Smad4 in renal inflammation and fibrosis were investigated in vivo and in vitro as described in the Chapter III, IV and V of this thesis. / Results: There were several novel findings through this thesis: / 1. TGF-β1 signals through its TβRII to diversely regulate renal fibrosis and inflammation. We found that disrupted TRII suppressed Smad3-dependent renal fibrosis while enhancing NF-κB-driven renal inflammation. Thus, TβRII not only acts as a binding receptor for initiating the TGF-β signaling, but also determines the diverse role of TGF-β1 in inflammation and fibrosis, which was described in the Chapter III. / 2. As shown in the Chapter IV, an unexpected finding from this thesis was that although Smad2 and Smad3 were homologically similar and bound together in response to TGF-β1 stimulation, Smad2 counter-regulated Smad3-mediated renal fibrosis. This was evidenced by the findings that conditional deletion of Smad2 enhanced Smad3 signaling including phosphorylation, nuclear translocation, the Smad3 responsive promoter activity, and the binding of Smad3 to Col1A2 promoter. Thus, disrupted Smad2 from the kidney significantly enhanced Smad3-mediated renal fibrosis in the UUO kidney and in cultured TEC. / 3. Finally, we also showed that that Smad4 acted not only as a common Smad in TGF-β signaling, but exerted its regulatory role in determining the diverse role of TGF-β1 in renal inflammation and fibrosis. Disruption of Smad4 significantly enhanced renal inflammation by impairing inhibitory effect of Smad7 on NF-κB-driven renal inflammation. In contrast, disrupted Smad4 inhibited renal fibrosis by blocking Smad3 functional activity without influencing Smad3 signaling. Because deletion of Smad4 inhibited TGF-β1-induced Smad3 responsive promoter activity and the binding of Smad3 to the Col1A2 promoter without altering the phosphorylation and nuclear translocation of Smad3 (Chapter V). / Conclusions: TβRII and Smad4 may function as key regulators of TGF-β signaling and diversely regulate the renal inflammation and fibrosis. Smad2 plays a protective role in renal fibrosis by counter-regulating Smad3 signaling. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Meng, Xiaoming. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 202-231). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.i / Declaration --- p.viii / Acknowledgement --- p.ix / Table of Contents --- p.xii / List of Abbreviations --- p.xxvii / List of Figures/Tables --- p.xxix / Chapter CHAPTER I --- INTRODUCTION --- p.1 / Chapter 1.1 --- TGF-β signaling pathway --- p.2 / Chapter 1.1.1 --- TGF-β superfamily --- p.2 / Chapter 1.1.2 --- TGF-β signaling transduction --- p.3 / Chapter 1.1.2.1 --- Smad-dependent TGF-β signaling --- p.4 / Chapter 1.1.2.2 --- Smad-independent TGF-β signaling --- p.10 / Chapter 1.2 --- Chronic Kideny disease (CKD) --- p.12 / Chapter 1.2.1 --- Epidemiology of CKD --- p.12 / Chapter 1.2.2 --- Pathophysiology of CKD --- p.12 / Chapter 1.3 --- TGF-β signaling in renal diseases --- p.13 / Chapter 1.3.1 --- Role of TGF-β1 in renal diseases --- p.13 / Chapter 1.3.2 --- Potential role of TβRII in renal diseases --- p.15 / Chapter 1.3.3 --- Potential role of Smad2 in renal diseases --- p.17 / Chapter 1.3.4 --- Potential role of Smad4 in renal diseases --- p.20 / Chapter 1.3.5 --- Role of Smad7 in renal diseases --- p.23 / Chapter 1.3.6 --- Role of Smad-independent TGF-β signaling in renal disease --- p.24 / Chapter CHAPTER II --- MATERIALS AND METHODS --- p.26 / Chapter 2.1 --- MATERIALS --- p.27 / Chapter 2.1.1 --- Reagents and Equipments --- p.27 / Chapter 2.1.1.1 --- General reagents and equipments for cell culture --- p.27 / Chapter 2.1.1.2 --- General reagents and equipments for real-time RT-PCR --- p.28 / Chapter 2.1.1.3 --- General reagents and equipments for Masson Trichrome Staining --- p.28 / Chapter 2.1.1.4 --- General reagents and equipments for Immunohistochemistry --- p.29 / Chapter 2.1.1.5 --- General reagents and equipments for Immunofluorescence --- p.29 / Chapter 2.1.1.6 --- General reagents and equipments for Western Blot --- p.29 / Chapter 2.1.1.7 --- General reagents and equipments for Promoter assay --- p.31 / Chapter 2.1.1.8 --- General reagents and equipments for ChIP assay --- p.32 / Chapter 2.1.2 --- Buffers --- p.32 / Chapter 2.1.2.1 --- Buffers for Immunohistochemistry --- p.32 / Chapter 2.1.2.2 --- Buffers for Western blot --- p.35 / Chapter 2.1.3 --- Sequences of Primers and siRNAs --- p.40 / Chapter 2.1.4 --- Antibodies --- p.42 / Chapter 2.2 --- METHODS --- p.44 / Chapter 2.2.1 --- Animal model of Unilateral Ureteral Obstruction (UUO) --- p.44 / Chapter 2.2.2 --- Cell culture --- p.44 / Chapter 2.2.2.1 --- NRK52E cell line --- p.44 / Chapter 2.2.2.2 --- Smad2 WT/KO mouse embryonic fibroblasts (MEFs) --- p.45 / Chapter 2.2.2.3 --- Primary culture of kidney fibroblasts --- p.45 / Chapter 2.2.2.4 --- Primary culture of peritoneal macrophages --- p.46 / Chapter 2.2.3 --- PAS staining --- p.47 / Chapter 2.2.3.1 --- Tissue Handling and Fixation --- p.47 / Chapter 2.2.3.2 --- Tissue embedding and sectioning --- p.47 / Chapter 2.2.3.3 --- Preparation of Paraffin Tissue Sections for PAS staining --- p.48 / Chapter 2.2.3.4 --- PAS staining --- p.48 / Chapter 2.2.4 --- Real-time RT-PCR --- p.48 / Chapter 2.2.4.1 --- Total RNA isolation --- p.48 / Chapter 2.2.4.2 --- Reverse Transcription --- p.49 / Chapter 2.2.4.3 --- Real-time PCR --- p.50 / Chapter 2.2.4.4 --- Analysis of Real-time PCR --- p.50 / Chapter 2.2.5 --- Masson Trichrome Staining --- p.51 / Chapter 2.2.6 --- Immunohistochemistry --- p.52 / Chapter 2.2.6.1 --- Preparation of Paraffin Tissue Sections for IHC --- p.52 / Chapter 2.2.6.2 --- Antigen-Antibody Reaction --- p.52 / Chapter 2.2.6.3 --- Signal Detection --- p.53 / Chapter 2.2.6.4 --- Semi-quantification of Immunohistochemistry --- p.53 / Chapter 2.2.7 --- Immunofluorescence --- p.54 / Chapter 2.2.8 --- Western blot analysis --- p.54 / Chapter 2.2.8.1 --- Protein preparation --- p.55 / Chapter 2.2.8.2 --- SDS-PAGE --- p.56 / Chapter 2.2.8.3 --- Transmembrane of protein --- p.56 / Chapter 2.2.8.4 --- Incubation of first and second antibody --- p.57 / Chapter 2.2.8.5 --- Signal capture and analysis --- p.57 / Chapter 2.2.8.6 --- Stripping --- p.57 / Chapter 2.2.9 --- Promoter assay --- p.58 / Chapter 2.2.10 --- ChIP assay --- p.61 / Chapter 2.2.11 --- Statistical analysis --- p.62 / Chapter CHAPTER III --- THE DIVERSE ROLE OF TGF-BETA RECEPTOR II IN RENAL INFLAMMATION AND FIBROSIS --- p.63 / Chapter 3.1 --- INTRODUCTION --- p.64 / Chapter 3.2 --- AIMS --- p.64 / Chapter 3.3 --- MATERIALS AND METHODS --- p.66 / Chapter 3.3.1 --- Generation and characterization of TβRII conditional Knockout mice --- p.66 / Chapter 3.3.2 --- Generation and characterization of TβRII disrupted tubular epithelial cell line (NRK52E) and kidney interstitial fibroblasts --- p.67 / Chapter 3.3.3 --- Animal model of Unilateral Ureteral Obstruction --- p.67 / Chapter 3.3.4 --- Cell culture --- p.67 / Chapter 3.3.5 --- Real-time RT-PCR --- p.68 / Chapter 3.3.6 --- Masson Trichrome Staining --- p.68 / Chapter 3.3.7 --- Immunohistochemistry --- p.68 / Chapter 3.3.8 --- PAS staining --- p.69 / Chapter 3.3.9 --- Immunofluorescence --- p.69 / Chapter 3.3.10 --- Western blot analysis --- p.70 / Chapter 3.3.11 --- Promoter assay --- p.70 / Chapter 3.3.12 --- Statistical analysis --- p.70 / Chapter 3.4 --- RESULTS --- p.71 / Chapter 3.4.1 --- Characterization of TβRII conditional Knockout mice and TβRII disrupted cells --- p.71 / Chapter 3.4.2 --- Disruption of TβRII suppresses renal interstitial damage in the UUO kidney --- p.72 / Chapter 3.4.3 --- Disruption of TβRII suppresses renal fibrosis in UUO kidney and TGF-β1-induced fibrotic response in vitro --- p.76 / Chapter 3.4.3.1 --- Conditional knockout of TβRII from the kidney decreases the collagen I level in UUO kidney --- p.76 / Chapter 3.4.3.2 --- Disruption of TβRII inhibits TGF-β1 induced collagen I level in vitro --- p.79 / Chapter 3.4.3.3 --- Conditional knockout of TβRII from the kidney decreases the α-SMA positive cells infiltration in vivo --- p.81 / Chapter 3.4.3.4 --- Disruption of TβRII inhibits TGF-β1-induced α-SMA expression in vitro --- p.83 / Chapter 3.4.3.5 --- Conditional knockout of TβRII from the kidney decreases the FN level in UUO nephropathy --- p.85 / Chapter 3.4.3.6 --- Disruption of TβRII decreases TGF-β1-induced FN expression in vitro --- p.87 / Chapter 3.4.4 --- Disruption of TβRII impairs the TGF-β/Smad signaling in vivo in the UUO kidney and in vitro in TGF-β1 treated tubular epithelial cells and kidney fibroblasts --- p.89 / Chapter 3.4.4.1 --- Conditional knockout of TβRII decreases the UUO induced TGF-β1 expression in vivo and the TGF-β1 auto-induction in vitro --- p.89 / Chapter 3.4.4.2 --- Disrupted TβRII decreases CTGF level in the UUO nephropathy in vivo and the TGF-β1 induced CTGF mRNA level in vitro --- p.91 / Chapter 3.4.4.3 --- Conditional knockout of TβRII impairs the Smad3 signaling in the injured kidney --- p.93 / Chapter 3.4.4.4 --- Disrupted TβRII inhibits TGF-β1-induced Smad3 phosphorylation, P-Smad3 nuclear translocation and Smad3 responsive promoter activity in vitro --- p.95 / Chapter 3.4.4.5 --- Conditional knockout of TβRII doesn’t alter the activation of ERK and P38 signaling in the UUO kidney --- p.97 / Chapter 3.4.4.6 --- Disrupted TβRII inhibits TGF-β1-induced ERK and P38 phosphorylation in vitro --- p.99 / Chapter 3.4.5 --- Disruption of TβRII enhances inflammatory cytokines expression in the UUO kidney and impairs the anti-inflammatory effect of TGF-β1 in response to IL-1β triggered inflammatory response in the TEC cells --- p.101 / Chapter 3.4.5.1 --- Conditional knockout of TβRII increases the TNF-α expression in the UUO nephropathy --- p.101 / Chapter 3.4.5.2 --- Conditional knockout of TβRII increases the IL-1β expression in the UUO nephropathy --- p.103 / Chapter 3.4.5.3 --- Conditional knockout of TβRII doesn’t enhance the MCP-1 expression and macrophages infiltration in the UUO nephropathy --- p.104 / Chapter 3.4.5.4 --- Disruption of TβRII in TECs decreases the anti-inflammatory effect of TGF-β1 in response to IL-1β --- p.106 / Chapter 3.4.6 --- Disruption of TβRII enhances NFκB activation in vivo and in vitro --- p.108 / Chapter 3.5 --- DISCUSSION --- p.110 / Chapter 3.6 --- CONCLUSION --- p.114 / Chapter CHAPTER IV --- Smad2 protects against TGF-β/Smad3 mediated renal fibrosis --- p.115 / Chapter 4.1 --- INTRODUCTION --- p.116 / Chapter 4.2 --- AIMS --- p.117 / Chapter 4.3 --- MATERIALS AND METHODS --- p.117 / Chapter 4.3.1 --- Generation and characterization of Smad2 conditional Knockout mice --- p.117 / Chapter 4.3.2 --- Generation and characterization of Smad2 KO MEFs and Smad2 knockdown/overexpression tubular epithelial cell line (NRK52E) --- p.118 / Chapter 4.3.3 --- Animal model of Unilateral Ureteral Obstruction --- p.118 / Chapter 4.3.4 --- Cell culture --- p.118 / Chapter 4.3.5 --- Real-time RT-PCR --- p.119 / Chapter 4.3.6 --- Western blot analysis --- p.119 / Chapter 4.3.7 --- Immunohistochemistry --- p.119 / Chapter 4.3.8 --- Masson Trichrome Staining --- p.119 / Chapter 4.3.9 --- Immunofluorescence --- p.120 / Chapter 4.3.10 --- Promoter assay --- p.120 / Chapter 4.3.11 --- ChIP assay --- p.120 / Chapter 4.3.12 --- Statistical analysis --- p.120 / Chapter 4.4 --- RESULTS --- p.121 / Chapter 4.4.1 --- Characterization of Smad2 disrupted mice and cells --- p.121 / Chapter 4.4.1.1 --- Characterization of Smad2 conditional Knockout mice --- p.121 / Chapter 4.4.1.2 --- Characterization of Smad2 knockout MEFs, Smad2 knockdown/overexpression TECs --- p.123 / Chapter 4.4.2 --- Disruption of Smad2 further enhances renal fibrosis in vivo and in vitro --- p.124 / Chapter 4.4.2.1 --- Conditional knockout of Smad2 increases total collagen deposition and Col.I level in the UUO kidney --- p.124 / Chapter 4.4.2.2 --- Disruption of Smad2 in MEFs and TECs increases Col.I production in a time- and dosage-dependent manner in response to TGF-β1 --- p.126 / Chapter 4.4.2.3 --- Conditional knockout of Smad2 increases Col.III level in the UUO kidney --- p.128 / Chapter 4.4.2.4 --- Disruption of Smad2 in MEFs and TECs increases Col.III production in a time- and dosage-dependent manner in response to TGF-β1 --- p.130 / Chapter 4.4.3 --- Disruption of Smad2 further enhances renal fibrosis by suppressing the collagen degradation system in vivo and in vitro --- p.132 / Chapter 4.4.3.1 --- Conditional knockout of Smad2 inhibits the MMP2 mRNA while enhances TIMP-1 production in UUO kidney --- p.132 / Chapter 4.4.3.2 --- Disruption of Smad2 in MEFs and TECs decreases the MMP2 level while enhances TIMP-1 production in response to TGF-β1 --- p.133 / Chapter 4.4.4 --- Disruption of Smad2 further increases renal fibrosis by increasing TGF-β1 auto-induction and CTGF level in vivo and in vitro --- p.135 / Chapter 4.4.4.1 --- Disruption of Smad2 increases TGF-β1 auto-induction in vivo and in vitro --- p.135 / Chapter 4.4.4.2 --- Disruption of Smad2 increases CTGF synthesis in vivo and in vitro --- p.137 / Chapter 4.4.5 --- Disruption of Smad2 further increases renal fibrosis by enhancing Smad3 signaling in vivo and in vitro --- p.139 / Chapter 4.4.5.1 --- Conditional knockout of Smad2 further enhances Smad3 phosphorylation and nuclear translocation --- p.139 / Chapter 4.4.5.2 --- Disruption of Smad2 in MEFs and TECs further enhances Smad3 phosphorylation, nuclear translocation, Smad3 responsive promoter activity and the binding to the Col1A2 promoter --- p.141 / Chapter 4.4.6 --- Overexpression of Smad2 suppresses Smad3 signaling therefore ameliorates the TGF-β1-induced fibrotic response in TECs --- p.144 / Chapter 4.4.6.1 --- Overexpression of Smad2 ameliorates the TGF-β1- induced fibrotic response in TECs --- p.144 / Chapter 4.4.6.2 --- Overexpression of Smad2 suppresses Smad3 phosphorylation --- p.146 / Chapter 4.5 --- DISCUSSION --- p.147 / Chapter 4.6 --- CONCLUSION --- p.150 / Chapter CHAPTER V --- THE DISTINCT ROLE OF SMAD4 IN RENAL INFLAMMATION AND FIBROSIS --- p.151 / Chapter 5.1 --- INTRODUCTION --- p.152 / Chapter 5.2 --- AIMS --- p.152 / Chapter 5.3 --- MATERIALS AND METHODS --- p.153 / Chapter 5.3.1 --- Generation and characterization of Smad4 conditional Knockout mice --- p.153 / Chapter 5.3.2 --- Generation and characterization of Smad4 disrupted kidney interstitial fibroblasts and peritoneal macrophages --- p.153 / Chapter 5.3.3 --- Animal model of Unilateral Ureteral Obstruction (UUO) --- p.154 / Chapter 5.3.4 --- Cell culture --- p.154 / Chapter 5.3.5 --- Real-time RT-PCR --- p.155 / Chapter 5.3.6 --- Western blot analysis --- p.155 / Chapter 5.3.7 --- Immunohistochemistry --- p.155 / Chapter 5.3.8 --- Masson Trichrome Staining --- p.155 / Chapter 5.3.9 --- Promoter assay --- p.156 / Chapter 5.3.10 --- ChIP assay --- p.156 / Chapter 5.3.11 --- Statistical analysis --- p.156 / Chapter 5.4 --- RESULTS --- p.157 / Chapter 5.4.1 --- Characterization of Smad4 conditional Knockout mice and Smad4 disrupted cells --- p.157 / Chapter 5.4.2 --- Disruption of Smad4 suppresses renal fibrosis in the UUO nephropathy in vivo and TGF-β1-induced fibrotic response in vitro --- p.160 / Chapter 5.4.2.1 --- Conditional knockout of Smad4 from the kidney decreases the total collagen deposition in the UUO nephropathy --- p.160 / Chapter 5.4.2.2 --- Conditional knockout of Smad4 from the kidney decreases the Col.I production in the UUO nephropathy --- p.161 / Chapter 5.4.2.3 --- Disruption of Smad4 inhibits TGF-β1-induced Col.I production in vitro --- p.163 / Chapter 5.4.3 --- Disruption of Smad4 impairs the Smad3 function in vivo and in vitro --- p.164 / Chapter 5.4.3.1 --- Conditional knockout of Smad4 doesn’t decrease Smad3 phosphorylation and P-Smad3 nuclear translocation in vivo and in vitro --- p.164 / Chapter 5.4.3.2 --- Disruption of Smad4 inhibits TGF-β1 induced Smad3 promoter activity and the Smad3 binding to Col1A2 promoter --- p.166 / Chapter 5.4.3.3 --- Disruption of Smad4 has minimal effect on the activation of ERK signaling in vivo and in vitro --- p.167 / Chapter 5.4.4 --- Disruption of Smad4 enhances renal inflammation and impairs the anti-inflammatory effect of TGF-β1 in response to IL-1β triggered inflammatory response in vitro --- p.169 / Chapter 5.4.4.1 --- Conditional knockout of Smad4 increases the inflammatory cells infiltration --- p.169 / Chapter 5.4.4.2 --- Conditional knockout of Smad4 increases the TNFα expression in the UUO nephropathy --- p.171 / Chapter 5.4.4.3 --- Conditional knockout of Smad4 increases the IL-1β expression in the UUO nephropathy --- p.172 / Chapter 5.4.4.4 --- Conditional knockout of Smad4 increases the MCP-1 expression in the UUO nephropathy --- p.173 / Chapter 5.4.4.5 --- Conditional knockout of Smad4 increases the ICAM-1 level in the UUO nephropathy --- p.174 / Chapter 5.4.4.6 --- Time and dosage dependent experiments in response to IL-1β in macrophages --- p.175 / Chapter 5.4.4.7 --- Disruption of Smad4 in macrophages decreases the anti-inflammatory effect of TGF-β1 in response to IL-1β --- p.176 / Chapter 5.4.5 --- Disruption of Smad4 impairs the inhibitory effect of Smad7 on NFκB activation in vivo and in vitro --- p.178 / Chapter 5.4.5.1 --- Conditional knockout of Smad4 largely inhibits Smad7 level in UUO kidney --- p.178 / Chapter 5.4.5.2 --- Conditional knockout of Smad4 suppresses IκBα and further increases NF-κB p65 activation in UUO kidney --- p.180 / Chapter 5.4.5.3 --- Disruption of Smad4 inhibits Smad7 synthesis in macrophages --- p.182 / Chapter 5.4.5.4 --- Conditional knockout of Smad4 impair the inhibition effect of TGF-β1 on the activation of NFκB p65 in macrophages --- p.184 / Chapter 5.5 --- DISCUSSION --- p.186 / Chapter 5.6 --- CONCLUSION --- p.189 / Chapter CHAPTER VI --- SUMMARY AND DISCUSSION OF THE MAJOR FINDINGS --- p.190 / Chapter 6.1 --- SUMMARY AND DISCUSSION --- p.192 / Chapter 6.1.1 --- The diverse role of TβRII in renal inflammation and fibrosis both in vivo and in vitro --- p.192 / Chapter 6.1.2 --- Smad2 protects renal fibrosis by counter-regulating Smad3 signaling --- p.192 / Chapter 6.1.3 --- Disruption of Smad4 increased renal inflammation while suppressed the renal fibrosis in vivo and in vitro --- p.194 / Chapter 6.1.4 --- Comparative analysis of functions and related mechanisms between TβRII and Smad4 in renal disease --- p.195 / Chapter 6.1.5 --- Inadequacies of current work and future plan --- p.197 / Chapter 6.1.6 --- Perspectives (1) : The balance within the TGF-b/Smad signaling may determine the fate of renal diseases --- p.197 / Chapter 6.1.7 --- Perspectives(2):The balance within the TGF-β/Smad signaling may determine the fate of renal diseases --- p.198 / Chapter 6.2 --- CONCLUSION --- p.201 / REFERENCES --- p.202 / PUBLICATION LIST --- p.232 / HONORS AND AWARDS --- p.237
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EFEITO DA LASERTERAPIA DE BAIXA INTENSIDADE NA MODULAÇÃO GÊNICA DE FATORES RELACIONADOS A CICATRIZAÇÃO DE ÚLCERAS DE PACIENTES DIABÉTICOSRuh, Anelice Calixto 03 December 2013 (has links)
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Previous issue date: 2013-12-03 / Pressure sores (PS) are wounds found mainly on bone surfaces where the tissue under pressure suffers ischemia which leads to cellular lesion. The causes of such lesion are several and its healing process depends on several factors, intrinsic and extrinsic to the individual. The objective of this study was to analyze the tissue expression of growth factors, IL6, TNF, VEGF, TGF, which take part in the tissue healing process under the effect of low-level laser InGaAIP. The study was approved by the Ethics Committee at UEPG and the individuals informed and made aware of the aims of the study. The group under study comprised 8 volunteers aged 30 to 75 years old, admitted at the Santa Casa de Misericórdia de Ponta Grossa (SCMPG) hospital, who presented sacroiliac PS, ranked as degree II, III or IV according to NPUAP (2009), both sexes. The PSs were photographed for classification and gross analysis of the lesion area through the program Image Pro Plus 6.0®. Samples of periulcerated tissue were obtained through biopsy before and after laser therapy. The biopsies were destined to the qRT-PCR (Quantitative Real Time PCR). Results revealed that in the gross evaluation of the wound there was improvement of the appearance and size of the lesion area, with increase in the granulation tissue. For the qRT-PCR tests, results point out a non-significant expression of differential growth factors between pre and post laser therapy condition. Based on these results it was possible to conclude that the laser therapy helps the healing process, but the expression of growth factors suffers influence of aspects that interfere in the healing process such as age, underlying pathology, medication, patient’s general condition, nutrition, and skin condition. / Úlceras por pressão (UPP) são feridas encontradas principalmente em superfícies ósseas aonde o tecido sob pressão sofre isquemia levando a lesão celular. As causas desta lesão são muitas e seu processo de cicatrização sofre influência de vários fatores, intrínsecos e extrínsecos do indivíduo. O objetivo desta pesquisa foi analisar a expressão tecidual dos fatores de crescimento, IL6, TNF, VEGF, TGF, que participam do processo de cicatrização tecidual sob os efeitos do laser de baixa potência InGaAIP. O trabalho foi aprovado pelo Comitê de Ética da UEPG e os indivíduos foram informados e esclarecidos sobre os objetivos da pesquisa. O grupo estudado foi composto de 8 voluntários com idade entre 30 a 75 anos, internados na Santa Casa de Misericórdia de Ponta Grossa (SCMPG), que apresentassem UPP sacroíliaca, classificadas em grau II, III ou IV segundo a NPUAP (2009), ambos os sexos. As UPP foram fotografadas para classificação e análise macroscópica da área da lesão através do programa Image Pro Plus 6.0®. Foram obtidas amostras do tecido peri-ulcerado através de biópsia antes e após aplicações de laserterapia. Estas biópsias foram destinadas aos ensaios de qRT-PCR (Quantitative Real Time PCR). Os resultados demonstram que na avaliação macroscópica da ferida houve melhora no aspecto e tamanho da área da lesão, com aumento do tecido de granulação. Para ensaios de qRT-PCR os resultados apontam uma expressão dos fatores de crescimento diferencial não significativa entre o estado pré e pós laserterapia. Com estes resultados foi possível concluir que a laserterapia ajuda no processo de cicatrização, mas que a expressão dos fatores de crescimento sofrem influência de aspectos que interferem no processo de cicatrização como, idade, patologias de base, medicamentos, estado geral paciente, nutrição, estado da pele.
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Marcadores da diferenciação osteoblástica em culturas de células crescidas sobre titânio e expostas a coquetel de fatores de crescimento e proteínas / Osteoblast differentiation markers in cultured cells grown on titanium and exposed to a cocktail of growth factors and proteinsMariana Sales de Melo Soares 24 July 2014 (has links)
Os efeitos de preparações de plasma rico em plaquetas (PRP) sobre a atividade osteogênica in vitro e in vivo em contato com biomateriais são divergentes na literatura. O objetivo do presente estudo foi avaliar a expressão e/ou atividade de marcadores iniciais da diferenciação osteoblástica em culturas de células osteogênicas crescidas sobre titânio (Ti) e expostas a coquetel de fatores de crescimento e proteínas PRP-símile (coquetel de FCs). Células osteoblásticas primárias derivadas de calvárias de ratos recém-nascidos foram cultivadas em meio osteogênico e expostas, nos 7 primeiros dias, a coquetel de FCs nas diluições 1:1 (FCs), 1:10 (FCs/10), 1:100 (FCs/100) e 1:1000 (FCs/1000). Foram avaliados, nos tempos de 7, 10 e 14 dias: 1) os aspectos morfológicos e a imunomarcação de sialoproteína óssea (BSP) e osteopontina (OPN), por epifluorescência; 2) a proliferação celular, por ensaio de MTT; 3) a expressão de RNAm para o fator de transcrição Runx2, BSP e fosfatase alcalina (ALP), por PCR em tempo real; 4) a atividade de ALP, clivada da fração de membrana; 5) quantificação da mineralização, por extração do vermelho de Alizarina. Os resultados mostraram inibição da formação dos nódulos de matriz mineralizada em culturas FCs e atraso em seu desenvolvimento em FCs/10 e FCs/100, em comparação a FCs/1000 e controle. A expressão de Runx2, BSP e ALP era menor em todas as culturas expostas ao coquetel de FCs em 7 dias, sendo que para Runx2 e BSP notava-se o efeito concentração-dependente em 10 dias. Menores valores de atividade de ALP foram observados nas culturas FCs e FCs/10, com efeito concentração-dependente e correlação positiva com a mineralização em 7 dias, mas não em 10 e 14. Os resultados permitem concluir que a exposição ao coquetel de FCs inibe e/ou atrasa a diferenciação osteogênica de culturas primárias sobre Ti. Adicionalmente, a atividade de ALP de membrana pode ser considerada também um marcador inicial de diferenciação osteoblástica, indicativo do potencial osteogênico no modelo in vitro utilizado. / The effects of platelet-rich plasma (PRP) preparations on the in vitro and in vivo osteogenic activity in contact with biomaterials have been subject of debate and controversy in the literature. The aim of the present study was to evaluate the expression and/or activity of early markers of osteoblast differentiation in cultured osteogenic cells grown on titanium (Ti) and exposed to a PRP-like cocktail of growth factors and proteins (GFs cocktail). Primary osteoblastic cells derived from newborn rat calvarial bone were cultured in an osteogenic medium (control group) and exposed during the first 7 days of culture to the following dilutions of GFs cocktail: 1:1 (GFs), 1:10 (GFs/10), 1:100 (GFs/100) and 1:1000 (GFs/1000). At days 7, 10 and 14 of culture, the following parameters were assessed: 1) morphology and immunolabeling for bone sialoprotein (BSP) and osteopontin (OPN) by epifluorescence microscopy; 2) cell proliferation by MTT assay; 3) mRNA expression for the osteoblast markers Runx2, BSP and alkaline phosphatase (ALP) by real time PCR; 4) ALP activity following ALP cleavage from cell membrane; 5) mineralization by Alizarin red extraction. The results showed no mineralized nodules for the GFs group and a delayed nodule formation for GFs/10 and GFs/100 compared with GFs/1000 and control. Whereas Runx2, BSP and ALP mRNA levels were lower for all cultures exposed to the GFs cocktail at day 7, a concentration-dependent effect was noticed only for Runx2 and BSP at day 10. The GFs cocktail showed a concentration-dependent effect on ALP activity, with the lowest values for GFs and GFs/10 cultures and a positive correlation with mineralization at day 7 but not at day 10 or 14. In conclusion, inhibited and/or delayed osteogenic differentiation take place in primary cultures grown on Ti and exposed to the GFs cocktail. In addition, membrane ALP activity can also be considered an early marker of osteoblast differentiation, indicative of the in vitro osteogenic potential in the model used.
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Rôles des cardiofibroblastes dans la protection des cardiomyocytes au cours de l'ischémie-reperfusion / Role of cardiac fibroblasts in cardiomyocyte protection during ischemia reperfusionAbrial, Maryline 07 November 2013 (has links)
Les cardiofibroblastes (CF) possèdent des rôles clés dans la régulation de la structure et du fonctionnement myocardique. Leurs implications physiopathologiques, notamment dans le remodelage et la fibrose, ont été largement décrites dans les maladies cardiovasculaires chroniques. Cependant, leurs rôles au cours de la phase aigüe d'ischémie-reperfusion (l/R) restent encore à élucider. Nous avons donc émis l'hypothèse que les CF pouvaient participer à la protection des cardiomyocytes (CM) face aux lésions d'l/R. Le but de ce travail a donc consisté en l'exploration et l'identification des mécanismes de cette protection. Un modèle cellulaire de CM et CF de rats nouveau-nés in vitro et un modèle d'l/R in vivo chez la souris ont été utilisés. Nos résultats montrent que la présence des CF, en co-culture avec les CM, augmente de façon paracrine leur viabilité, face aux lésions d'l/R. Cette action paracrine a été confirmée par l'utilisation du sécrétome de CF hypoxiques capable, à lui seul, d'augmenter la viabilité des CM. Ces résultats ont été corroborés par des expériences d'l/R in vivo, dans lesquelles les souris traitées avec le sécrétome de CF présentent une diminution de la taille d'infarctus. De plus, nous avons montré que TlMP-1, un facteur fortement détecté dans le sécrétome de CF, est capable de diminuer à la fois la mortalité cellulaire in vitro des CM et la taille de l'infarctus in vivo. L'utilisation d'inhibiteurs pharmacologiques nous a permis de mettre en évidence que cette protection paracrine était médiée en partie par l'activation des voies de signalisation Pl3K/Akt et ERK1/2. En conclusion cette étude démontre pour la première fois que les CF participent, de façon paracrine, à la protection des CM au cours la phase aigüe d'ischémie reperfusion. TlMP-1 semble être un des facteurs clé de cette cardioprotection par les CF. En parallèle de ce travail, plusieurs études collaboratives ont été réalisées, sur une cible majeure d'investigation dans la cardioprotection : le pore de transition de perméabilité mitochondriale et notamment sa régulation par le complexe l de la chaîne respiratoire et les échanges calciques, ainsi que son implication dans la défaillance multi-organe face à l'arrêt cardiaque / Roles of cardiac fibroblasts (CF) in the regulation of myocardial structure and function have been emphasized in the last decade. Their implications in pathophysiological aspects of chronic heart diseases such as myocardial remodelling and fibrosis is now well established. However their contribution to the acute phase of ischemia reperfusion injury still remains elusive. We hypothesized that CF may contribute to cardiomyocytes (CM) protection against ischemia reperfusion (l/R) injuries. This study was designed to investigate this protection and identify some of its mechanisms. Experiments were performed both on isolated neonatal rat CF and CM in vitro and in vivo mice model of myocardial infarction. We demonstrated that the presence of CF increases CM viability in co-cultures and that CF protect CM against l/R injuries in a paracrine manner. lt was confirmed by a similar effect of hypoxic CF secretome alone on CM viability. These findings were corroborated by in vivo experiments in which an infarct size reduction was observed in CF secretome treated mice. Furthermore, experiments with Tissue lnhibitor of Metalloproteinases-1 (TlMP-1), abundantly detected in CF secretome, was able to both decrease CM cell death and infarct size. Experiments with pharmacological inhibitors provided more evidence that this paracrine protection is partly mediated by Pl3K/Akt and ERK signalling pathways. Our data demonstrated for the first time that CF participate in cardioprotection during the acute phase of ischemia reperfusion, via a paracrine pathway, involving TlMP-1. Besides this first work, other collaborative studies have been performed, to investigate a major target in cardioprotection research : the mitochondrial permeability transition pore and its regulation by chain respiratory complex l and Ca2+ transfers and finally its implication in multiple organ failure in cardiac arrest
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Innovation thérapeutique dans la dysfonction endothéliale associée à l'hypertension artérielle pulmonaire / Novel molecular targets for correcting endothelial dysfunction associated with pulmonary arterial hypertensionTu, Ly 05 July 2012 (has links)
L’hypertension artérielle pulmonaire (HTAP) est une maladie grave caractérisée par une obstruction progressive des petites artères pulmonaires, conduisant à une augmentation progressive des résistances vasculaires pulmonaires (RVP) et à terme à une défaillance cardiaque droite et au décès du patient. La vasoconstriction, le remodelage vasculaire pulmonaire, la thrombose in situ et la dysfonction endothéliale sont autant de facteurs qui contribuent au développement et à la progression de la maladie. Ces travaux de doctorat ont visé à préciser et mieux comprendre le phénotype anormal de la cellule endothéliale (CE) pulmonaire de patients HTAP afin d’identifier de nouvelles cibles thérapeutiques adéquates pour corriger et restaurer la dysfonction endothéliale associée à l’hypertension pulmonaire. Plusieurs perturbations fonctionnelles liées à des anomalies moléculaires ont pu être identifiées. Les CE HTAPi sont caractérisées par une prolifération, une migration, une survie et une réponse excessive à différents facteurs de croissance : PDGF, EGF, et FGF2. Parmi les anomalies moléculaires à l’origine de ces perturbations fonctionnelles, nous avons retrouvé une production et une libération anormale de FGF2, de MMP2, de MIF, d’IL-6 et de MCP-1, mais aussi une activité constitutive de la MAPK, une surexpression des facteurs anti-apoptotiques BCL2 et BCL-xL, de FGFR2, de p130cas et de CD74. Cette meilleure compréhension de la CE-HTAPi a déjà permis d’identifier plusieurs cibles qui pourraient être évaluées et à aussi permis de soulever un certain nombre de questions qui requiert des études plus poussées. / Pulmonary arterial hypertension (PAH) is a severe disease characterized by progressive obstruction of small pulmonary arteries, leading to a progressive increase in pulmonary vascular resistance and to right heart failure and death. Vasoconstriction, pulmonary vascular remodeling, in situ thrombosis and endothelial dysfunction are all factors that contribute to the development and progression of the disease. These work aimed to clarify and better understand the abnormal phenotype of the pulmonary endothelial cell (P-EC) of PAH patients to identify new therapeutic targets to correct and restore the endothelial dysfunction associated with pulmonary hypertension. Several functional disturbances related to molecular abnormalities have been identified. The IPAH-P-EC is characterized by proliferation, migration, survival and an excessive response to various growth factors: PDGF, EGF, and FGF2. Among the molecular abnormalities responsible for these functional disturbances, we found an abnormal production and release of FGF2, MMP2, MIF, IL-6 and MCP-1, but also a constitutive activity of MAPK, overexpression of anti-apoptotic factors BCL2 and BCL-xL, of FGFR2, of p130Cas and CD74. This better understanding of the EC-IPAH has already identified several targets that could be evaluated and also helped raise a number of issues requiring further study.
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Marcadores da diferenciação osteoblástica em culturas de células crescidas sobre titânio e expostas a coquetel de fatores de crescimento e proteínas / Osteoblast differentiation markers in cultured cells grown on titanium and exposed to a cocktail of growth factors and proteinsSoares, Mariana Sales de Melo 24 July 2014 (has links)
Os efeitos de preparações de plasma rico em plaquetas (PRP) sobre a atividade osteogênica in vitro e in vivo em contato com biomateriais são divergentes na literatura. O objetivo do presente estudo foi avaliar a expressão e/ou atividade de marcadores iniciais da diferenciação osteoblástica em culturas de células osteogênicas crescidas sobre titânio (Ti) e expostas a coquetel de fatores de crescimento e proteínas PRP-símile (coquetel de FCs). Células osteoblásticas primárias derivadas de calvárias de ratos recém-nascidos foram cultivadas em meio osteogênico e expostas, nos 7 primeiros dias, a coquetel de FCs nas diluições 1:1 (FCs), 1:10 (FCs/10), 1:100 (FCs/100) e 1:1000 (FCs/1000). Foram avaliados, nos tempos de 7, 10 e 14 dias: 1) os aspectos morfológicos e a imunomarcação de sialoproteína óssea (BSP) e osteopontina (OPN), por epifluorescência; 2) a proliferação celular, por ensaio de MTT; 3) a expressão de RNAm para o fator de transcrição Runx2, BSP e fosfatase alcalina (ALP), por PCR em tempo real; 4) a atividade de ALP, clivada da fração de membrana; 5) quantificação da mineralização, por extração do vermelho de Alizarina. Os resultados mostraram inibição da formação dos nódulos de matriz mineralizada em culturas FCs e atraso em seu desenvolvimento em FCs/10 e FCs/100, em comparação a FCs/1000 e controle. A expressão de Runx2, BSP e ALP era menor em todas as culturas expostas ao coquetel de FCs em 7 dias, sendo que para Runx2 e BSP notava-se o efeito concentração-dependente em 10 dias. Menores valores de atividade de ALP foram observados nas culturas FCs e FCs/10, com efeito concentração-dependente e correlação positiva com a mineralização em 7 dias, mas não em 10 e 14. Os resultados permitem concluir que a exposição ao coquetel de FCs inibe e/ou atrasa a diferenciação osteogênica de culturas primárias sobre Ti. Adicionalmente, a atividade de ALP de membrana pode ser considerada também um marcador inicial de diferenciação osteoblástica, indicativo do potencial osteogênico no modelo in vitro utilizado. / The effects of platelet-rich plasma (PRP) preparations on the in vitro and in vivo osteogenic activity in contact with biomaterials have been subject of debate and controversy in the literature. The aim of the present study was to evaluate the expression and/or activity of early markers of osteoblast differentiation in cultured osteogenic cells grown on titanium (Ti) and exposed to a PRP-like cocktail of growth factors and proteins (GFs cocktail). Primary osteoblastic cells derived from newborn rat calvarial bone were cultured in an osteogenic medium (control group) and exposed during the first 7 days of culture to the following dilutions of GFs cocktail: 1:1 (GFs), 1:10 (GFs/10), 1:100 (GFs/100) and 1:1000 (GFs/1000). At days 7, 10 and 14 of culture, the following parameters were assessed: 1) morphology and immunolabeling for bone sialoprotein (BSP) and osteopontin (OPN) by epifluorescence microscopy; 2) cell proliferation by MTT assay; 3) mRNA expression for the osteoblast markers Runx2, BSP and alkaline phosphatase (ALP) by real time PCR; 4) ALP activity following ALP cleavage from cell membrane; 5) mineralization by Alizarin red extraction. The results showed no mineralized nodules for the GFs group and a delayed nodule formation for GFs/10 and GFs/100 compared with GFs/1000 and control. Whereas Runx2, BSP and ALP mRNA levels were lower for all cultures exposed to the GFs cocktail at day 7, a concentration-dependent effect was noticed only for Runx2 and BSP at day 10. The GFs cocktail showed a concentration-dependent effect on ALP activity, with the lowest values for GFs and GFs/10 cultures and a positive correlation with mineralization at day 7 but not at day 10 or 14. In conclusion, inhibited and/or delayed osteogenic differentiation take place in primary cultures grown on Ti and exposed to the GFs cocktail. In addition, membrane ALP activity can also be considered an early marker of osteoblast differentiation, indicative of the in vitro osteogenic potential in the model used.
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Développement d'implants actifs et nanostructurés à des fins de régénération dentaire / Development of active and nanostructured implants for dental regenerationBécavin, Thibault 17 December 2015 (has links)
L’ingénierie tissulaire a vu émerger, dans la dernière décennie, la nanomédecinerégénérative combinant non seulement les cellules souches mais aussi les facteursde croissance. Le but de ce travail a été d’utiliser les techniques de l’ingénierieosseuse et pulpaire pour améliorer ou créer des implants actifs et vivants.L’ingénierie tissulaire vise à remplacer ou à réparer des tissus endommagés del’organisme par des implants bioactifs imitant les tissus naturels. Ainsi, il est nécessairede développer en premier lieu des matériaux biocompatibles, c’est-à-direacceptés par l’organisme, et capables d’interagir avec les cellules au site del’implantation du dit matériau afin d’aider à la fonction de l’implant. Pour cette raison,de nombreuses méthodes visent le développement de biomatériaux implantablesimitant le mieux possible la matrice extracellulaire. Dans cette étude,l’"électrospinning" a été utilisé afin de concevoir des membranes nanofibreuses. Ellesont ensuite été fonctionnalisées et associées à des germes de 1re molaire mandibulairede souris afin d'en favoriser le développement. Après une revue de littératures'intéressant à l'ingénierie tissulaire en chirurgie dentaire, deux axes de rechercheseront développés. Le premier concerne la régénération de l’unité os/dent et le deuxièmel'innervation de la dent. / In the last decade, regenerative nanomedicine emerged from tissue engineeringby combining stem cells and growth factors. The aim of this work was to use thebone and dental pulp tissue engineering technics to improve or create new living andactive implants. Tissue engineering aims to replace or repair damaged tissues withbioactive implants mimicking natural tissue. Thus, it is necessary to develop in a firstplace, new biocompatible materials. These materials should be well accepted by theorganism and capable to interact with the cells present in the implantation site to helpthe implant integration. In this study, Electrospinning was used to create nanofibrousmembrane which size and organization are comparable to the extracellular matrix.These membranes were functionalized and associated with first molar embryologicalgerms to promote their development. After a literature review about tissue engineeringin dental surgery, two research thematics will be developed. The first thematic isto regenerate the bone/tooth unit and the second is to innervate the tooth.
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Compara??o entre protocolos para obten??o de plasma rico em plaquetas em c?es: estudo celular / Comparison between protocols for obtaining platelet-rich plasma in dogs: a cellular studyVIDAL JUNIOR, Andr? William Masseaux 15 February 2017 (has links)
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2017 - Andr? William Masseaux Vidal J?nior.pdf: 1275550 bytes, checksum: d2d211215832b8ab2e53ab8d6da5ae4b (MD5)
Previous issue date: 2017-02-15 / It was proposed by this study to evaluate two protocols (PA and PB) to obtain autologous canine PRP, which is easy to perform in an ambulatory (semiautomatic method) and of good quality (platelet concentration, qualitative evaluation of platelet morphology and low contamination with Erythrocytes), to later propose different therapeutic indications of these PRPs as tissue modulating agents, according to the observed cellular leukocyte pattern. For this purpose, 20 dogs (Canis lupus familiaris) were used at the UFRRJ (HV) Veterinary Hospital, males and females, aged between 1 and 7 years (mean 4 years), considered clinically and hematologically healthy for elective surgeries and / or routine consultations. After adequate trichotomy and antisepsis, 8 mL of blood were collected by venipuncture of the jugular, being immediately packed in two 4 mL flasks, vacuntainer type, containing sodium citrate 3,2%. Protocol A using double centrifugation with 210 xG and 370 xG and protocol B using double centrifugation with 140 x G and 330 x G. PRP samples obtained from each protocol were used to count platelets, erythrocytes and leukocytes in the Neubauer chamber, differential leukocyte counting and observation of platelet morphology in smears. Data (mean and standard deviations) were analyzed by the 95% probability t test (p <0,05) using Pearson's correlation to test the relationship between platelet and erythrocyte counts, platelets and leukocytes and leukocytes in Relation to red blood cells. There was a very weak negative correlation between platelets and leukocytes (? = -0,03), weak negative between platelets and erythrocytes (? = -0,3) and strong positive correlation between leukocytes and erythrocytes (? = 0,75). Although Protocol B did not reach the desired one million platelets average (979300 ? 79631 cells / ?L), both protocols, A and B (4,42 ? 1,61 and 3,85 ? 1,55 times more platelets than Total blood, respectively) (p <0,05) were efficient in concentrating platelets. The cytoplasmic prolongations evidencing platelet activation were present in 26,55 ? 6,72% of platelets of protocol A and 26,25 ? 7,03% in those of protocol B (p> 0,05). A and B presented a small number of red blood cells (p> 0,05), which were considered to be contaminants of the samples and, for the quantity of leukocytes, protocol A presented more white blood cells (p <0,05) than protocol B with higher concentrations of basophils , and lymphocytes. / Foi proposto por esse estudo avaliar dois protocolos (PA e PB) para obten??o de PRP canino aut?logo, de f?cil execu??o em ambulat?rio (m?todo semiautom?tico) e de boa qualidade (capacidade de concentra??o de plaquetas, avalia??o qualitativa da morfologia das plaquetas e reduzida contamina??o com eritr?citos), para posteriormente propor diferentes indica??es terap?uticas desses PRP como agentes moduladores de recupera??o tecidual, de acordo com o padr?o celular leucocit?rio observado. Para isso foram utilizados 20 c?es (Canis lupus familiaris) atendidos no Hospital Veterin?rio da UFRRJ (HV), machos e f?meas, com idade variando entre 1 e 7 anos (m?dia 4 anos), considerados saud?veis clinica e hematologicamente que se destinavam para cirurgias eletivas e/ou consultas de rotina. Ap?s tricotomia e antissepsia adequadas, eram coletados 8 mL de sangue por venopun??o da jugular sendo imediatamente acondicionados em dois frascos de 4 mL, do tipo vacuntainer, contendo citrato de s?dio a 3,2%. O protocolo A utilizando centrifuga??o dupla com 210 xG e 370 xG e protocolo B utilizando centrifuga??o dupla com 140 xG e 330 xG. Amostras de PRP obtidas a partir de cada protocolo foram destinadas a contagem de plaquetas, hem?cias e leuc?citos em c?mara de Neubauer, contagem diferencial dos leuc?citos e observa??o da morfologia das plaquetas em esfrega?os. Analisou-se os dados (m?dias e desvios padr?o) pelo Teste t com 95% de probabilidade (p<0,05) utilizando-se correla??o de Pearson para testar a rela??o entre a contagem de plaquetas e hem?cias, plaquetas e leuc?citos e leuc?citos em rela??o as hem?cias. Houve correla??o negativa muito fraca entre plaquetas e leuc?citos (?= -0,03), negativa fraca entre plaquetas e hem?cias (?= -0,3) e correla??o positiva forte entre leuc?citos e hem?cias (?=0,75). Embora o protocolo B n?o tenha alcan?ando a m?dia um milh?o de plaquetas desejado (979300 ? 79631 c?lulas/?L), ambos os protocolos, A e B (4,42 ? 1,61 e 3,85 ? 1,55 vezes mais plaquetas que o sangue total, respectivamente) (p<0,05) foram eficientes em concentrar plaquetas. Os prolongamentos citoplasm?ticos evidenciando ativa??o plaquet?ria estiveram presentes em 26,55 ? 6,72 % das plaquetas do protocolo A e 26,25 ? 7,03 % nas do protocolo B (p>0,05). PA e PB apresentaram reduzido n?mero de hem?cias (p>0,05) consideradas contaminantes das amostras e, quanto a quantidade de leuc?citos, o protocolo A apresentou mais gl?bulos brancos (p<0,05) que o protocolo B com maiores concentra??es de bas?filos, segmentados e linf?citos.
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An investigation of the effect of nerve growth factor in the early stages of neuronal differentiation.January 2007 (has links)
Yung, Him Shun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 133-146). / Abstracts in English and Chinese. / Abstract --- p.i / 論文摘要 --- p.iv / Acknowledgements --- p.vi / Publications based on work in this thesis --- p.vii / Abbreviations --- p.viii / Contents --- p.xi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Objectives and overview of this study --- p.1 / Chapter 1.2 --- Rat pheochromocytoma (PC12) cells --- p.3 / Chapter 1.3 --- Prostanoids and their receptors --- p.4 / Chapter 1.4 --- Roles of prostanoids --- p.7 / Chapter 1.5 --- Nerve growth factor (NGF) and its receptors --- p.9 / Chapter 1.6 --- Change of gene expressions by NGF in PC12 cells --- p.10 / Chapter 1.7 --- Signaling pathways involved in NGF-induced differentiation of PC12 cells --- p.12 / Chapter 1.8 --- Classification of adenylyl cyclases --- p.14 / Chapter 1.9 --- Methods to study differentiation of PCI 2 cells --- p.15 / Chapter Chapter 2 --- Materials and Methods --- p.19 / Chapter 2.1 --- Materials --- p.19 / Chapter 2.2 --- Cell culture medium and buffers --- p.25 / Chapter 2.3 --- Buffers and solutions for assay of [3H]inositoI phosphates ([3H]IP) production --- p.25 / Chapter 2.4 --- Buffers and solutions for assay of [3H]cAMP production --- p.27 / Chapter 2.5 --- Buffers and solutions for Western blotting --- p.28 / Chapter 2.6 --- Methods --- p.30 / Chapter 2.6.1 --- Maintenance of PC12 cells --- p.30 / Chapter 2.6.2 --- General culture condition of PCI2 cells for NGF treatment --- p.31 / Chapter 2.6.3 --- Determination of phospholipase C activity in PC12 cells --- p.31 / Chapter 2.6.3.1 --- Principle of assay --- p.31 / Chapter 2.6.3.2 --- Column preparation --- p.32 / Chapter 2.6.3.3 --- Measurement of [3H]IP production --- p.33 / Chapter 2.6.3.4 --- Data analysis --- p.34 / Chapter 2.6.4 --- Determination of adenylyl cyclase activity in PC12 cells --- p.35 / Chapter 2.6.4.1 --- Principle of assay --- p.35 / Chapter 2.6.4.2 --- Column preparation --- p.35 / Chapter 2.6.4.3 --- Measurement of [3H]cAMP production --- p.36 / Chapter 2.6.4.4 --- Data analysis --- p.37 / Chapter 2.6.5 --- Determination of neurofilament protein expression in PC12 cells by Western blotting --- p.38 / Chapter 2.6.6 --- Determination of adenylyl cyclase isoform expression in PC12 cells by reverse transcriptase-polymerase chain reaction (RT-PCR) --- p.39 / Chapter 2.6.6.1 --- Isolation of total cellular RNA --- p.39 / Chapter 2.6.6.2 --- Synthesis of first strand cDNA by reverse transcription (RT) --- p.40 / Chapter 2.6.6.3 --- Polymerase Chain Reaction (PCR) --- p.41 / Chapter 2.6.6.4 --- Agarose gel electrophoresis --- p.41 / Chapter 2.6.7 --- Neurite quantification --- p.42 / Chapter 2.6.8 --- Trypan blue exclusion test --- p.42 / Chapter Chapter 3 --- Results --- p.45 / Chapter 3.1 --- Characterization of prostanoid receptor expression in PC12 cells . --- p.45 / Chapter 3.1.1 --- Study of the presence of Gq-coupled prostanoid receptors --- p.45 / Chapter 3.1.2 --- Study of the presence of Gs-co»pled prostanoid receptors --- p.47 / Chapter 3.1.3 --- Study of the presence of Gi-coupled prostanoid receptors --- p.48 / Chapter 3.1.4 --- Further proof of EP3 expression in PC12 cells --- p.50 / Chapter 3.1.5 --- Discussion --- p.51 / Chapter 3.2 --- Time course effect of NGF on PC12 cells --- p.65 / Chapter 3.2.1 --- Effect of NGF on PGE2-mediated inhibition of forskolin-stimulated [3H]cAMP production --- p.65 / Chapter 3.2.2 --- Effect of NGF on basal and forskolin-stimulated [3H]cAMP production --- p.67 / Chapter 3.2.3 --- Acute effect of NGF on [3H]cAMP production --- p.70 / Chapter 3.2.4 --- Effect of NGF withdrawal on basal and forskolin-stimulated [3H]cAMP production --- p.71 / Chapter 3.2.5 --- Effect of NGF on adenylyl cyclase gene expression --- p.72 / Chapter 3.2.6 --- Discussion --- p.74 / Chapter 3.3 --- Quantification of the degree of differentiation of PC12 cells --- p.89 / Chapter 3.3.1 --- Expression of neurofilament protein as a marker of differentiation --- p.89 / Chapter 3.3.2 --- Neurite assays --- p.90 / Chapter 3.3.2.1 --- Manual assessment of PC12 cells --- p.90 / Chapter 3.3.2.2 --- Quantification of images of PC1 2 cells --- p.91 / Chapter 3.3.3 --- Discussion --- p.93 / Chapter 3.4 --- Adenosine A2a receptor activity in PC12 cells --- p.106 / Chapter 3.4.1 --- Effect of NGF on A2Areceptor-mediated [3H]cAMP production --- p.106 / Chapter 3.4.2 --- Synergistic activation of adenylyl cyclase by A2A receptor and forskolin --- p.108 / Chapter 3.4.3 --- Chronic and acute effect of ADA and ZM241385 on [3H]cAMP production --- p.109 / Chapter 3.4.3.1 --- Chronic effect of ADA and ZM241385 --- p.110 / Chapter 3.4.3.2 --- Acute effect of ADA and ZM241385 --- p.111 / Chapter 3.4.4 --- Discussion --- p.112 / Chapter Chapter 4 --- Discussion and future perspectives --- p.121 / Chapter 4.1 --- Discussion --- p.121 / Chapter 4.2 --- Future perspectives --- p.131 / References --- p.133
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