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The role of osteocyte Kindlin-2 in the anabolic actions of PTH in boneFu, Xuekun 01 May 2020 (has links)
In vertebrates, PTH receptor 1 (PTH1R) plays a pivotal role in control of bone development and homeostasis; however, how it is regulated is poorly defined. Here we report that Kindlin-2 binds to and modulates PTH1R to regulate bone mass and PTH actions. Deleting Kindlin-2 expression using the 10-kb mouse Dmp1-Cre severely impairs the anabolic effects of intermittent PTH on bone in adult mice with or without ovariectomy. Of particular interest, Kindlin-2 and Pth1r double heterozygous mice (Dmp1- Cre; Kindlin-2 f/+ ; Pth1r f/+ ), but not either singly heterozygous mice (Dmp1- Cre; Kindlin-2 f/+ or Dmp1-Cre; Pth1r f/+ ), display severe osteopenia and fail to increase bone mass in response to administration of intermittent PTH. Mechanistically, Kindlin-2 interacts with the C-terminal cytoplasmic region of PTH1R. When overexpressed, this region efficiently inhibits the endogenous PTH/PTH1R signaling in osteoblasts, which is reversed by introduction of a point mutation that abolishes the Kindlin-2 interaction. Furthermore, Kindlin-2 loss inhibits PTH-induced CREB phosphorylation and cAMP production in vitro and in bone. PTH upregulates, while estrogen deficiency downregulates, expression of Kindlin-2 in vitro and in bone. Collectively, we demonstrate that interplay between Kindlin-2 and PTH1R regulates bone mass by modulating PTH1R and provide a potential therapeutic target for metabolic bone diseases
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The effect of statins on bone and mineral metabolismMaritz, Frans Jacobus 04 1900 (has links)
Dissertation (PhD)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: The Effect of Statins on Bone and Mineral Metabolism
Both statins and amino-bisphosphonates reduce the prenylation of proteins which
are involved in cytoskeletal organization and activation of polarized and motile cells.
Consequently statins have been postulated to affect bone metabolism. We investigated
the effects of different doses of simvastatin (1,5,10 and 20mg/Kg/day), administered orally
over 12 weeks to intact female Sprague-Dawley rats, and the effect of simvastatin
20mg/Kg/day in sham and ovariectomised rats, on femoral bone mineral density (BMD)
and quantitative bone histomorphometry (QBH), compared to controls. Similarly, the affect
of atorvastatin (2,5mg/Kg/day) and pravastatin (10mg/Kg/day) on BMD was investigated
and compared to controls. BMD was decreased by simvastatin 1mg/Kg/day (p = 0.042),
atorvastatin (p = 0,0002) and pravastatin (p = 0.002). The effect on QBH parameters
differed with different doses of simvastatin (ANOVA; p = 0.00012). QBH parameters of
both bone formation and resorption were equivalently and markedly increased by
simvastatin 20mg/Kg/day in two independent groups of intact rats, and reflected by a
relatively unchanged BMD. At lower doses, simvastatin 1mg/Kg/day decreased bone
formation while increasing bone resorption as reflected by a marked decrease in BMD.
Ovariectomised animals receiving simvastatin 20mg/Kg/day showed no change in BMD
relative to the untreated ovariectomised controls, their increase in bone formation was
smaller than in sham-operated rats receiving simvastatin and there was no change in
bone resorption. The dose response curves of simvastatin for bone formation and
resorption differed from each other.
From these studies it is concluded that:-
a) low-dose simvastatin (1mg/Kg/day), atorvastatin 2.5mg/Kg/day) and pravastatin
10mg/Kg/day) decrease BMD in rodents;b) 1mg/Kg/day simvastatin decreases bone formation and increases bone
resorption and is reflected by a reduced BMD;
c) 20mg/Kg/day simvastatin increases bone formation and resorption and results
in an unchanged BMD;
d) the effects of simvastatin on QBH differ at different dosages;
e) the dose-response curves for QBH parameters of bone resorption and bone
formation differ from each other;
f) the effects of simvastatin seen in intact rats are not observed in ovariectomised
rats;
g) simvastatin is unable to prevent the bone loss caused by ovariectomy. / AFRIKAANSE OPSOMMING: Die Effek van Statiene op Been en Mineraal Metabolisme
Beide statiene en aminobisfosfonate verminder die prenelasie van proteïene wat
betrokke is in die sitoskeletale organisasie en aktivering van gepolariseerde en
beweeglike selle. Gevolglik is dit gepostuleer dat statiene ‘n invloed sal hê op been
metabolisme. Ons het die effekte van verskillende dossisse van simvastatien (1, 5, 10 en
20mg/Kg/dag), mondelings toegedien oor 12 weke aan intakte vroulike Sprague-Dawley
rotte, en die effek van simvastatien 20mg/Kg/dag op skyn- en ge-ovariektomeerde rotte,
op femorale been mineral digtheid (BMD) en kwantitatiewe been histomorfometrie (KBH),
vergeleke met kontroles, ondersoek. Op ‘n soortgelyke manier is die effek van
atorvastatien (2,5mg/Kg/day) en pravastatien (10mgKg/dag) op BMD ondersoek en
vergelyk met kontroles. BMD is verminder deur simvastatien 1mg/Kg/dag (p = 0.042),
atorvastatien (p = 0.0002) en pravastatien (p = 0.002). Die effekte op KBH parameters het
verskil met verskillende dossisse van simvastatien (ANOVA; p = 0.00012). KBH
parameters van beide been vormasie en resorpsie is vergelykend en merkbaar verhoog
deur simvastatien 20mg/Kg/dag in twee onafhanklike groepe van intakte rotte en is
vergesel deur ‘n relatiewe onveranderde BMD. Met laer dossisse het simvastatien
1mg/Kg/dag been vormasie verminder terwyl been resorpsie verhoog is en is weerspieël
deur ‘n merkbaar verminderde BMD. Ge-ovariektomeerde diere wat simvastatien
20mg/Kg/dag ontvang het, het geen verandering in BMD relatief tot die onbehandelde geovariektomeerde
kontroles getoon nie, en die toename in been vormasie was kleiner as in
die skyngeopereerde rotte wat simvastatien ontvang het en daar was geen verandering in
been resorpsie nie. Die dosis-respons kurwes vir simvastatien vir been vormasie en
resorpsie het van mekaar verskil.
Uit hierdie studies word die volgende gevolgtrekkings gea) lae-dosis simvastatien (1mg/Kg/dag), atorvastatien 2.5mg/Kg/dag en
pravastatien 10mg/Kg/dag verminder BMD in knaagdiere;
b) 1mg/Kg/dag simvastatien verminder been vormasie en verhoog been resorpsie
en veroorsaak gevolglik ‘n velaging in die BMD;
c) 20mg/Kg/dag simvastatien verhoog been vormasie en resorpsie met ‘n
gevolglike onveranderde BMD;
d) die effekte van simvastatien op KBH verskil met verskillende dossisse;
e) die dosis-repons kurwes van been resorpsie en been vormasie veskil van
mekaar
f) die effekte van simvastatien wat waargeneem in intakte rotte word nie gesien in
ge-ovariektomeerde rotte nie;
g) simvastatien kannie die verlies van been wat veroorsaak word deur
ovariektomie voorkom nie.
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Novel insights into the mechanistic gene regulation of STAT3 in bone cellsCorry, Kylie A. 25 June 2015 (has links)
Indiana University-Purdue University Indianapolis (IUPUI) / Many cells are involved in the orchestra that is bone homeostasis--particularly osteoclasts and osteoblasts, which mediate remodeling of bones. This creates a balance that must be kept in check, otherwise pathologies arise. The JAK-STAT signaling pathway is crucial to maintaining this balance. It has long been known that the transcription factor STAT3 has more profound effects on bone homeostasis than other members of the STAT family of proteins. Recently, a genetic condition called Job’s Syndrome has been specifically linked to point mutations in the Stat3 gene. These patients present with severe bone abnormalities, including prominent foreheads, broad nasal bridges, and abnormal eye spacing. For this reason, our lab has extensively studied conditional knockouts of Stat3 in all three types of bones cells in mice and observed severe deficiencies in numerous parameters of normal bone phenotypes. STAT3 seems to play a principal role in the signaling that takes place upon mechanical loading of bone tissues and calling cells into action where they are needed. Furthermore,
STAT3 has been found to be up-regulated in the early-response gene cluster following mechanical loading.
Our current approach to studying STAT3’s effects on bone includes both in vivo and in vitro comparisons of WT and KO STAT3 models. The conditional knock-out of STAT3 in 8-week old mice revealed significant phenotypic variations as compared to the WT controls, while no significant differences were observed in cKO newborn pups. We also looked at immortalized WT and STAT3 KO cell lines. The STAT3 KO cells had diminished proliferation rates and decreased differentiation capabilities. Furthermore, STAT3 KO cells showed significantly reduced mRNA levels of both Wnt3a and Wnt5a when exposed to fluid shear stress.
By employing available ChIP-seq data, we were able to elucidate the genome-wide binding patterns of STAT3. From the peak distribution, we can begin to uncover novel downstream effectors of STAT3 signaling that are responsible for the observed phenotypes in our conditional knockout mouse model. A preliminary look at the ChIP-seq data reveals Wnt and Nrf2 signaling to be under the putative control of STAT3. In our further research, we endeavor to experimentally confirm the ChIP-seq data for STAT3 with RNA-seq experiments in the hopes of finding potential therapeutic targets for bone pathologies.
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Development of siRNA delivery systems for approaching bone formation surfaces and for targeting osteoblasts.January 2012 (has links)
目前,骨形成低下的骨代謝異常在臨床中面臨巨大挑戰。治療這些疾病的途徑之一可通過小干擾核酸沉默骨形成抑制的基因。隨著核酸干擾技術的快速發展,採用核酸干擾策略進行治療的很多問題已被解決。然而,小干擾核酸的安全和有效遞送仍然是核酸干擾治療進行臨床轉化的瓶頸。其主要問題在於促進骨形成治療所需的小干擾核酸劑量較大,其系統給藥後可能對其他非骨組織產生副作用。所以,亟需針對具有促進成骨潛力的小干擾核酸開發安全有效的遞送系統。本研究的目的就是針對具有促進成骨潛力的小干擾核酸開發特定的遞送系統,以便應用於核酸干擾治療中的促進骨形成。策略之一是利用靶向骨形成表面的遞送系統攜載小干擾核酸到富集于骨形成表面的成骨系細胞。策略之二是直接把小干擾核酸遞送到成骨細胞,使其具有高度的細胞選擇性。在該研究中,我們採用具有成骨潛能的酪蛋白激酶2相互作用蛋白1小干擾核酸作為模型小干擾核酸以考察基因沉默效率。 / 靶向骨形成表面的(天門冬氨酸-絲氨酸-絲氨酸)₆-脂質體-小干擾核酸遞送系統:首先對多肽序列(天門冬氨酸-絲氨酸-絲氨酸)₆靶向骨形成表面的特性進行鑒定。進一步將(天門冬氨酸-絲氨酸-絲氨酸)₆作為靶向分子與以DOTAP為主要成分的陽離子脂質體進行連接製備(天門冬氨酸-絲氨酸-絲氨酸)6-脂質體遞送系統。採用凍幹/再水化方法對小干擾核酸進行包裹並對其粒徑,ζ電位,包封率以及穩定性進行考察。最後分別在體外和體內模型對該遞送系統遞送效果以及其攜載小干擾核酸的基因沉默效率進行評價。 / 實驗結果證實(天門冬氨酸-絲氨酸-絲氨酸)₆是一種在體內可以有效靶向骨形成表面的多肽。(天門冬氨酸-絲氨酸-絲氨酸)₆-脂質體的平均粒徑為140 nm左右,其包封率可高達80%。該遞送系統較穩定,可使攜載的小干擾核酸具有較高的基因沉默效率,而且沒有明顯的細胞毒性。體內試驗表明,該遞送系統在促進小干擾核酸在骨組織的分佈同時降低其被肝組織的攝取。該遞送系統所攜帶的酪蛋白激酶2相互作用蛋白1小干擾核酸可選擇性地沉默骨組織中的酪蛋白激酶2相互作用蛋白1基因,且對其他組織並沒有明顯影響。該結果表明(天門冬氨酸-絲氨酸-絲氨酸)₆-脂質體可促進小干擾核酸靶向骨組織並在骨組織沉默攜載小干擾核酸相應的基因。免疫化學分析結果顯示(天門冬氨酸-絲氨酸-絲氨酸)₆-脂質體可攜載小干擾核酸選擇性地到達骨形成表面的成骨系細胞,避免被前破骨細胞/破骨細胞吞噬。大鼠骨髓細胞採用Alp,Stro-1和Oscar抗體分選後的酪蛋白激酶2相互作用蛋白1 mRNA表達水平顯示該遞送系統可選擇性地沉默成骨系細胞。 / 靶向成骨細胞的L6適配子-脂質納米顆粒-小干擾核酸遞送系統:將針對大鼠成骨細胞(ROS 17/2.8細胞系)進行正向篩選,大鼠肝細胞(BRL-3A細胞系)和外周血細胞進行負向篩選的L6適配子與以DLin-KC2-DMA為主要成分的脂質納米顆粒採用膠束形式插入的方法進行連接製備L6適配子-脂質納米顆粒-小干擾核酸遞送系統。並對其粒徑,ζ電位,包封率和形態學進行考察。在體外評價實驗中,考察了該遞送系統的選擇性,細胞毒性,基因沉默效率以及細胞攝取機制。在體內實驗中,對小干擾核酸的組織分佈以及其攜載小干擾核酸在成骨細胞和肝細胞的分佈進行了評價。 / 實驗結果顯示L6適配子-脂質納米顆粒-小干擾核酸的平均粒徑為84.0±5.3 nm,其電勢為-23 ± 2 mV,包封率為80.8 ± 3.4%. 脂質納米顆粒表面的L6適配子可促進小干擾核酸在ROS 17/2.8細胞系(靶向細胞)中的攝取, 然而在BRL-3A 細胞系(非靶向細胞)中攝入很少。該遞送系統沒有明顯細胞毒性,在10 nM小干擾核酸的低濃度下,體外基因沉默效率可高達50 % 以上。由L6適配子引起的巨胞被證實是成骨細胞攝取L6適配子-脂質納米顆粒所攜載小干擾核酸的主要機制。體內實驗顯示該遞送系統可促進小干擾核酸在骨組織的分佈,降低其被肝組織的攝取。在肝组织冰凍切片中,肝血竇和肝細胞中沒有明顯的小干擾核酸分佈,進一步說明該遞送系統可降低對肝組織的影響。免疫化學分析結果顯示L6適配子-脂質納米顆粒-小干擾核酸可攜載小干擾核酸選擇性地到達成骨細胞,避免被前破骨細胞/破骨細胞吞噬。 / 重要意義:本研究中的兩種新型小干擾核酸系統可分別選擇性地遞送小干擾核酸靶向骨形成表面和成骨細胞。 (天門冬氨酸-絲氨酸-絲氨酸)₆-脂質體-小干擾核酸遞送系統開拓了全新的途徑,實現選擇性地遞送小干擾核酸到骨形成表面從而降低對骨吸收的影響。 L6適配子-脂質納米顆粒-小干擾核酸遞送系統在成骨細胞表面特徵蛋白未知的情況下,首次採用適配子技術在細胞水準實現成骨細胞的選擇性遞送。該研究中的兩種遞送系統為核酸干擾治療的促進骨形成策略提供了強而有力的工具,為實現肌肉骨骼疾病相關領域的核酸干擾治療策略從基礎科學向臨床應用的轉化建立了堅實的基礎。 / Metabolic skeletal disorders that are associated with impaired bone formation are a major clinical challenge. One approach to treat these diseases was to silence bone formation-inhibitory genes by small interference RNAs (siRNAs). With the rapid development of RNA interference (RNAi) technology, more issues of RNAi-based therapy strategies have been addressed. However, the safe and effective delivery of siRNAs is still the bottleneck for its translation from bench to bedside. One major concern was that the large therapeutic doses of systemically administered siRNA to stimulate sufficient bone formation may carry a high risk for adverse effects on non-skeletal tissues. Therefore, development of specific siRNA delivery systems for safe and efficient transporting osteogenic siRNAs is highly desirable. The objective of the present study was to explore siRNA delivery systems for osteogenic siRNAs in RNAi-based bone anabolic therapy. One strategy was to develop siRNA delivery system targeting bone formation surfaces to facilitate delivery of siRNAs to osteogenic cells. Another approch was to develop siRNA delivery system targeting osteoblasts directly. Plekho1 siRNA targeting casein kinase-2 interacting protein-1 (Ckip-1) with osteogenic potential was employed as a representative siRNA in our current study. / (AspSerSer)6-liposome-siRNA for targeting bone formation surfaces: (AspSerSer)6 for targeting bone formation surfaces was firstly identified. Then, (AspSerSer)6 was conjugated with DOTAP-based liposome to produce (AspSerSer)6-liposome. (AspSerSer)6-liposome-siNRA was prepared by lyophilization/rehydration method and characterized in terms of particle size, zeta potential, encapsulation efficiency and the stability in serum. Finally, the delivery of siRNA and the corresponding gene silencing mediated by (AspSerSer)6-liposome-siRNA were evaluated in the in vitro and in vivo models. / The results indicated that the novel (AspSerSer)₆ was a promising peptide for targeting bone formation surfaces in vivo. (AspSerSer)₆-liposome with the average particle size of 140 nm encapsulating Plekho1 siRNA exhibited more than 80% encapsulation efficiency and good stability against enzymatic degradation. It demonstrated high knockdown efficiency without obvious cytotoxicity. In in vivo study, the result of tissue distribution experiment indicated that (AspSerSer)6-liposome-siRNA enhanced the distribution of siRNA in bone, meanwhile reduced the uptake of siRNA in liver. The Plekho1 protein and mRNA expression in various tissues demonstrated that (AspSerSer)₆-liposome-siRNA could facilitate gene silencing in a bone-selective manner. The results of immunochemistry analyses indicated (AspSerSer)₆-liposome-siRNA facilitated delivering siRNA to osteogenic cells at bone formation surfaces and avoided siRNA to pre-osteoclast/osteoclast. Plekho1 mRNA expression in rat bone marrow cells sorted by fluorescence activated cell sorting (FACS) using Alp, Stro-1 and Oscar antibody, respectively, further suggested (AspSerSer)₆-liposome-siRNA could silence gene in a cell-selective manner in vivo. / L6-LNPs-siRNA for targeting osteoblasts: L6 aptamer for targeting osteoblasts (ROS 17/2.8 cell line) and using rat hepatocyte (BRL-3A cell line) and peripheral blood cells in negative selection was conjugated to DLin-KC2-DMA-based lipid nanoparticles (LNPs) to generate L6-LNPs-siRNA by post-insertion method in the form of micelles. L6-LNPs-siRNA was characterized with particle size, zeta potential, encapsulation efficiency and morphology. Its selectivity, cytotoxicity and knockdown efficiency were evaluated in vitro. The mechanism of L6-LNPs-mediated siRNA cellular uptake was further investigated. The tissue distribution of the injected siRNA and the localization of the siRNA with osteoblasts as well as hepatocytes were also evaluated in vivo. / The results showed L6-LNPs-siRNA have the average particle size of 84.0 ± 5.3 nm and zeta potential of -23 ± 2 mV. Its encapsulation efficiency was 80.8 ± 3.4%. The L6 aptamer on the surface of LNPs facilitated the cellular uptake of Plekho1 siRNA in ROS 17/2.8 cell line (target cells) but no uptake in BRL-3A cell line (non-target cells) in vitro. L6-LNPs-siRNA with low cytotoxicity exhibited above 50% knockdown efficiency at a low concentration of 10 nM in vitro. Macropinocytosis induced by L6 was demonstrated to be the predominant mechanism of L6-LNPs mediated siRNA uptake in osteoblasts. In in vivo study, it was shown that L6-LNPs-siRNA facilitated the distribution of siRNA in bone and decreased the hepatic uptake. No obvious siRNA fluorescent signals in sinus and hepatocyte was observed in liver cryosection further indicated the reducing influence on liver after administration of L6-LNPs-siRNA. Co-localization of fluorescence-labeled siRNA with Alp-positive cells was dominantly documented, whereas there were no instances of such overlapping staining with Oscar-positive cells after L6-LNPs-siRNA treatment, which suggested L6-LNPs-siRNA facilitated delivering siRNA in a cell-selective manner in vivo. / Significance: These two innovative siRNA delivery systems in the present study selectively targeted bone formation surfaces and osteoblasts, respectively. (AspSerSer)₆-liposome-siRNA opened up a new avenue to specifically deliver therapeutic siRNAs to bone formation surfaces without affecting bone resorption. L6-LNPs-siRNA achieved the osteoblast-specific delivery for siRNA at cellular level by aptamer technology for the first time, even without knowledge of characteristic protein on the surface of osteoblasts. The two delivery systems provided the powerful tools for RNAi-based bone anabolic strategy and established a solid foundation for translating RNAi-based therapies from basic science to clinic applications in the musculoskeletal field. / 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. / Wu, Heng. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 130-142). / Abstract also in Chinese. / Acknowledgements --- p.i / Abstract --- p.iii / 論文摘要 --- p.vi / Table of contents --- p.ix / Publications --- p.xiv / List of tables --- p.xvi / List of figures --- p.xvii / List of abbreviations --- p.xxi / Chapter One Introduction --- p.1 / Chapter 1.1 --- Great challenges in skeletal disorders --- p.2 / Chapter 1.2 --- RNA interference (RNAi) as therapeutic strategy --- p.3 / Chapter 1.2.1 --- Mechanism of RNAi --- p.3 / Chapter 1.2.2 --- Potential triggers of RNAi-mediated gene silencing --- p.4 / Chapter 1.2.3 --- Current clinical trials using RNAi as therapeutic strategy --- p.7 / Chapter 1.2.4 --- Current application of therapeutic siRNAs in skeletal disorders --- p.11 / Chapter 1.3 --- Challenges of siRNA in vivo delivery for targeting bone --- p.12 / Chapter 1.3.1 --- General challenges of siRNA delivery in vivo --- p.13 / Chapter 1.3.2 --- Challenges of siRNA delivery to bone --- p.15 / Chapter 1.3.2.1 --- Physiological property --- p.15 / Chapter 1.3.2.2 --- Targeting ligands for approaching bone --- p.16 / Chapter 1.4 --- Strategies of siRNAs in vivo delivery after systemic administration --- p.18 / Chapter 1.4.1 --- Naked siRNA and naked siRNA with chemical conjugation --- p.18 / Chapter 1.4.2 --- Nanoparticle delivery systems --- p.20 / Chapter 1.4.2.1 --- Liposome and lipid-like materials --- p.20 / Chapter 1.4.2.2 --- Polymers --- p.22 / Chapter 1.4.2.3 --- Targeted delivery system --- p.23 / Chapter 1.5 --- Strategies of osteogenic siRNAs delivery for stimulating bone formation --- p.24 / Chapter 1.6 --- Objective of present study --- p.25 / Chapter Chapter Two --- Preparation and characterization of (AspSerSer)₆-liposome-siRNA for targeting bone formation surfaces --- p.26 / Chapter 2.1 --- Introduction --- p.27 / Chapter 2.2 --- Materials and Methods --- p.28 / Chapter 2.2.1 --- Materials --- p.28 / Chapter 2.2.2 --- Identification of (AspSerSer)₆ --- p.29 / Chapter 2.2.3 --- Development of formulation --- p.30 / Chapter 2.2.3.1 --- Selection of the molar ratio of DOTAP --- p.30 / Chapter 2.2.3.2 --- Selection of the molar ratio of siRNA to lipids --- p.30 / Chapter 2.2.4 --- Preparation of (AspSerSer)6-liposome-siRNA --- p.30 / Chapter 2.2.5 --- Characterization of (AspSerSer)₆-liposome --- p.33 / Chapter 2.2.5.1 --- Particle Size and Zeta Potential --- p.33 / Chapter 2.2.5.2 --- Encapsulation Efficiency --- p.33 / Chapter 2.2.5.3 --- Stability in serum --- p.33 / Chapter 2.3 --- Results --- p.34 / Chapter 2.3.1 --- (AspSerSer)₆ as a targeting moiety --- p.34 / Chapter 2.3.2 --- Development of formulation --- p.37 / Chapter 2.3.3 --- Particle size, Zeta Potential and Encapsulation Efficiency --- p.38 / Chapter 2.3.4 --- Stability in serum --- p.38 / Chapter 2.4 --- Discussion --- p.40 / Chapter 2.5 --- Conclusion --- p.42 / Chapter Chapter Three --- Evaluation of (AspSerSer)₆-liposome-siRNA for cell-specific delivery and gene silencing in vitro and in vivo --- p.43 / Chapter 3.1 --- Introduction --- p.44 / Chapter 3.2 --- Materials and Methods --- p.45 / Chapter 3.2.1 --- Materials --- p.45 / Chapter 3.2.2 --- Biological evaluation in vitro --- p.46 / Chapter 3.2.2.1 --- Binding affinity with hydroxyapatite --- p.46 / Chapter 3.2.2.2 --- Cell culture --- p.46 / Chapter 3.2.2.3 --- Cellular uptake --- p.47 / Chapter 3.2.2.4 --- Knockdown efficiency in vitro --- p.47 / Chapter 3.2.2.5 --- Total RNA extraction, reverse transcription and quantitative real-time PCR --- p.48 / Chapter 3.2.3 --- Cytotoxicity --- p.49 / Chapter 3.2.4 --- Tissue distribution --- p.50 / Chapter 3.2.4.1 --- Experimental design --- p.50 / Chapter 3.2.4.2 --- Fluorescence image analysis --- p.50 / Chapter 3.2.4.3 --- Quantitative Analysis --- p.50 / Chapter 3.2.5 --- Localization of siRNA in liver --- p.51 / Chapter 3.2.5.1 --- Experimental design --- p.51 / Chapter 3.2.5.2 --- Histochemisty analysis --- p.51 / Chapter 3.2.6 --- Gene silencing in tissues --- p.52 / Chapter 3.2.6.1 --- Experimental design --- p.52 / Chapter 3.2.6.2 --- Determination of mRNA expression --- p.52 / Chapter 3.2.6.3 --- Western blot analysis --- p.52 / Chapter 3.2.7 --- Localization of siRNA with Osteoblasts/Osteoclasts --- p.53 / Chapter 3.2.7.1 --- Experimental design --- p.53 / Chapter 3.2.7.2 --- Immunohistochemistry analysis --- p.53 / Chapter 3.2.8 --- Gene silencing at cellular levels --- p.54 / Chapter 3.2.8.1 --- Experimental design --- p.54 / Chapter 3.2.8.2 --- Flow cytometry cell sorting --- p.54 / Chapter 3.2.9 --- Statistical analysis --- p.55 / Chapter 3.3 --- Results --- p.56 / Chapter 3.3.1 --- Binding affinity with hydroxyapatite --- p.56 / Chapter 3.3.2 --- Cellular uptake --- p.57 / Chapter 3.3.3 --- Knockdown efficiency in vitro --- p.57 / Chapter 3.3.4 --- Cytotoxicity --- p.59 / Chapter 3.3.5 --- Tissue distribution by imaging analysis --- p.60 / Chapter 3.3.6 --- Quantitative analysis of tissue distribution --- p.62 / Chapter 3.3.7 --- Localization of siRNA in liver --- p.63 / Chapter 3.3.8 --- Plekho1 mRNA and protein expressions --- p.64 / Chapter 3.3.9 --- Immunohistochemistry analysis --- p.65 / Chapter 3.3.10 --- Gene silencing at cellular level --- p.71 / Chapter 3.4 --- Discussion --- p.74 / Chapter 3.5 --- Conclusion --- p.77 / Chapter Chapter Four --- Preparation and characterization of aptamer-functionalized lipid nanoparticle for siRNA cell-specific delivery --- p.78 / Chapter 4.1 --- Introduction --- p.79 / Chapter 4.2 --- Materials and Methods --- p.80 / Chapter 4.2.1 --- Materials --- p.80 / Chapter 4.2.2 --- Synthesis of 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-di- oxolane (DLin-KC2-DMA) --- p.80 / Chapter 4.2.2.1 --- Synthesis of Linoleyl alcohol (1) --- p.81 / Chapter 4.2.2.2 --- Synthesis of Linoleyl bromide (2) --- p.81 / Chapter 4.2.2.3 --- Synthesis of Dilinoleylmethyl formate (3) --- p.82 / Chapter 4.2.2.4 --- Synthesis of Dilinoleyl Methanol (4) --- p.82 / Chapter 4.2.2.5 --- Synthesis of Dilinoleyl Ketone (5) --- p.83 / Chapter 4.2.2.6 --- Synthesis of 2, 2- Dilinoleyl- 4- (2-hydroxyethyl)-[1,3]-dioxolane (6) --- p.83 / Chapter 4.2.2.7 --- Synthesis of DLin-KC2-DMA --- p.83 / Chapter 4.2.3 --- Development of formulation --- p.84 / Chapter 4.2.3.1 --- Selection of the molar ratio of lipids --- p.84 / Chapter 4.2.3.2 --- Selection of the mass ratios of siRNA to lipids --- p.85 / Chapter 4.2.3.3 --- Selection of the molar ratios of L6-PEG2000-DSPE on L6-LNPs-siRNA --- p.85 / Chapter 4.2.4 --- Binding affinity with osteoblasts --- p.86 / Chapter 4.2.5 --- Preparation of L6-LNPs-siRNA --- p.86 / Chapter 4.2.5.1 --- Synthesis of L6-PEG2000-DSPE --- p.87 / Chapter 4.2.5.2 --- Preparation of LNPs-siRNA --- p.87 / Chapter 4.2.5.3 --- Post-insertion of aptamers on the surface of LNPs-siRNA --- p.88 / Chapter 4.2.6 --- Characterization of L6-LNPs-siRNA --- p.88 / Chapter 4.2.6.1 --- Particle size and Zeta Potential --- p.88 / Chapter 4.2.6.2 --- Encapsulation Efficiency (EE) --- p.88 / Chapter 4.2.6.3 --- Cryo-Transmission electron microscope --- p.89 / Chapter 4.3 --- Results --- p.90 / Chapter 4.3.1 --- Synthesis of DLin-KC2-DMA --- p.90 / Chapter 4.3.2 --- Formulation development --- p.93 / Chapter 4.3.3 --- Preparation of L6-LNPs --- p.95 / Chapter 4.3.4 --- Characterization of L6-LNPs-siRNA --- p.96 / Chapter 4.4 --- Discussion --- p.98 / Chapter 4.5 --- Conclusion --- p.101 / Chapter Chapter Five --- Evaluation of L6 aptamer functionalized lipid nanoparticles (L6-LNPs-siRNA) for osteoblast-specific delivery in vitro and in vivo --- p.102 / Chapter 5.1 --- Introduction --- p.103 / Chapter 5.2 --- Materials and Methods --- p.103 / Chapter 5.2.1 --- Materials --- p.103 / Chapter 5.2.2 --- Biological evaluation in vitro --- p.104 / Chapter 5.2.2.1 --- Cell culture --- p.104 / Chapter 5.2.2.2 --- Binding affinity with target/non-target cells --- p.105 / Chapter 5.2.2.3 --- Cellular uptake of siRNA in target/non-target cells --- p.105 / Chapter 5.2.2.4 --- Knockdown efficiency in vitro --- p.105 / Chapter 5.2.3 --- Cytotoxicity --- p.106 / Chapter 5.2.4 --- Mechanism of cellular uptake --- p.106 / Chapter 5.2.4.1 --- Spectral bio-imaging for endocytic pathways --- p.106 / Chapter 5.2.4.2 --- Chemical inhibition for endocytic pathways --- p.107 / Chapter 5.2.4.3 --- Determination of membrane ruffling --- p.107 / Chapter 5.2.5 --- Evaluation of specific delivery in vivo --- p.107 / Chapter 5.2.5.1 --- Experimental design --- p.107 / Chapter 5.2.5.2 --- Tissue distribution --- p.108 / Chapter 5.2.5.3 --- Localization of siRNA in liver --- p.108 / Chapter 5.2.5.4 --- Localization of siRNA with osteoblast/osteoclast --- p.108 / Chapter 5.2.6 --- Statistical analysis --- p.109 / Chapter 5.3 --- Results --- p.109 / Chapter 5.3.1 --- Binding selectivity of L6-LNPs-siRNA --- p.109 / Chapter 5.3.2 --- Selectivity of siRNA cellular uptake --- p.111 / Chapter 5.3.3 --- Knockdown efficiency in vitro --- p.112 / Chapter 5.3.4 --- Cytotoxicity --- p.113 / Chapter 5.3.5 --- Mechanism of cellular uptake --- p.113 / Chapter 5.3.6 --- Tissue distribution --- p.118 / Chapter 5.3.7 --- Localization of siRNA in liver --- p.119 / Chapter 5.3.8 --- Localization of siRNA with Osteoblasts/Osteoclasts --- p.120 / Chapter 5.4 --- Discussion --- p.123 / Chapter 5.5 --- Conclusion --- p.125 / Chapter Chapter Six --- Summary of the study and future research --- p.126 / Chapter 6.1 --- Summary of the study --- p.127 / Chapter 6.2 --- Future research --- p.128 / References --- p.130
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Bone Metabolism: The Role of STAT3 and Reactive Oxygen SpeciesNewnum, America Bethanne 14 August 2013 (has links)
Indiana University-Purdue University Indianapolis (IUPUI) / Signal Transducers and Activators of Transcription 3 (STAT3), a transcription factor expressed in many cell types, including osteoblasts and osteoclasts, is emerging as a key regulator of bone mass and strength. STAT3 mutations cause a rare human immunodeficiency disease characterized by extremely elevated levels of IgE in serum that have associated craniofacial and skeletal features, such as reduced bone mineral density and recurrent pathological fractures. Our microarray data and immunohistochemical staining using a normal rat model have shown that STAT3 mRNA and protein levels markedly increase in response to mechanical loading. In addition, as indicated by STAT3 phosphorylation in MC3T3-E1 osteoblastic cells, STAT3 activity significantly increases in response to 30 to 90 minutes fluid shear stress. In order to further study the role that STAT3 plays in bone responsiveness to loading, tissue-selective STAT3 knockout (KO) mice, in which inactivation of STAT3 occurs in osteoblasts, were generated by breeding the transgenic mice in which Cre recombinase cDNA was cloned downstream of a 3.6 or 2.3 kb fragment of the rat Col1a1 promoter (Col3.6-Cre and Col2.3-Cre, respectively) with a strain of floxed mice in which the two loxP sites flank exons 18-20 of the STAT3 gene were used. Mice engineered with bone selective inactivation of STAT3 in osteoblasts exhibited significantly lower bone mineral density (7-12%, p<0.05) and reduced ultimate force (21-34%, p<0.01) compared to their age-matched littermate controls. The right ulnae of 16-week-old bone specific STAT3 KO mice and the age-matched control mice were loaded with peak forces of 2.5 N and 2.75 N for female and male mice, respectively, at 2 Hz, 120 cycles/day for 3 consecutive days. Mice with inactivation of STAT3 specific in bone were significantly less responsive to mechanical loading than the control mice as indicated by decreased relative mineralizing surface (rMS/BS, 47-59%, p<0.05) and relative bone formation rate (rBFR/BS, 64-75%, p<0.001). Bone responsiveness was equally decreased in mice in which STAT3 is inactivated either in early osteoblasts (Col3.6-Cre) or in mature osteoblasts (Col2.3-Cre).
Accumulating evidence indicates that bone metabolism is significantly affected by activities in mitochondria. For instance, although STAT3 is reported to be involved in bone formation and resorption through regulation of nuclear genes, inactivation of STAT3 is shown to disrupt mitochondrial activities and result in an increased level of reactive oxygen species (ROS). Inactivation of STAT3 suppressed load-driven mitochondrial activity, which led to an elevated level of ROS in cultured primary osteoblasts. Oxidative stress induced by administration of buthionine sulfoximine (BSO) significantly inhibits load-induced bone formation in wild type mice. Taken together, the results support the notion that the loss-of-function mutation of STAT3 in osteoblasts and osteocytes diminishes load-driven bone formation and impairs the regulation of oxidative stress in mitochondria.
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The role of STAT3 in osteoclast mediated bone resorptionHimes, Evan 01 August 2014 (has links)
Indiana University-Purdue University Indianapolis (IUPUI) / Signal Transducer and Activator of Transcription 3 (STAT3) is known to be related to bone metabolism. Mutation of STAT3 causes a rare disorder in which serum levels of IgE are elevated. This causes various skeletal problems similar to osteoporosis.
To examine the effect of STAT3 in the osteoclast, we obtained two osteoclast specific STAT3 knockout mouse models: one using the CTSK promoter to drive Cre recombinase and another using a TRAP promoter. Examination of these mice at 8 weeks of age revealed a decreased trabecular bone volume in CTSK specific STAT3 knockout mice along with a slight decrease in osteoclast number in both CTSK and TRAP specific STAT3 knockout females. We also noticed changes in bone mineral density and bone mechanical strength in females. These data suggest that STAT3 plays a part in the function of the osteoclast.
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The essential role of Stat3 in bone homeostasis and mechanotransductionZhou, Hongkang January 2014 (has links)
Indiana University-Purdue University Indianapolis (IUPUI) / Signal Transducer and Activator of Transcription 3 (Stat3) is a transcription factor expressed in bone and joint cells that include osteoblasts, osteocytes, osteoclasts, and chondrocytes. Stat3 is activated by a variety of cytokines and growth factors, including IL-6/gp130 family cytokines. These cytokines not only regulate the differentiation of osteoblasts and osteoclasts, but also regulate proliferation of chondrocytes through Stat3 activation. In 2007, mutations of Stat3 have been confirmed to cause a rare human immunodeficiency disease – Job syndrome which presents skeletal abnormalities like: reduced bone density (osteopenia), scoliosis, hyperextensibility of joints, and recurrent pathological bone fractures. Changes in the Stat3 gene alter the structure and function of the Stat3 proteins, impairing its ability to control the activity of other genes. However, little is known about the effects of Stat3 mutations on bone cells and tissues.
To investigate the in vivo physiological role of Stat3 in bone homeostasis, osteoblast/osteocyte-specific Stat3 knockout (KO) mice were generated via the Cre-LoxP recombination system. The osteoblast/osteocyte-specific Stat3 KO mice showed bone abnormalities and an osteoporotic phenotype because of a reduced bone formation rate.
Furthermore, inactivation of Stat3 decreased load-driven bone formation, and the disruption of Stat3 in osteoblasts suppressed load-driven mitochondrial activity, which led to an elevated level of reactive oxygen species (ROS) in cultured primary osteoblasts.
Stat3 has been found to be responsive to mechanical stimulation, and might play an important role in mechanical signal transduction in osteocytes. To investigate the role Stat3 plays in mechanical signaling transduction, osteocyte-specific Stat3 knockout (KO) mice were created. Inactivation of Stat3 in osteocytes presented a significantly reduced load-driven bone formation. Decreased osteoblast activity indicated by reduced osteoid surface was also found in osteocyte-specific Stat3 KO mice. Moreover, sclerostin (SOST) protein which is a critical osteocyte-specific inhibitor of bone formation, its encoded gene SOST expression has been found to be enhanced in osteocyte-specific Stat3 KO mice.
Thus, these results clearly demonstrated that Stat3 plays an important role in bone homeostasis and mechanotransduction, and Stat3 is not only involved in bone-formation-important genes regulation in the nucleus but also in mediation of ROS and oxidative stress in mitochondria.
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