Biochemical and mechanical properties of the extracellular matrix (ECM) are known to independently influence cell function. Given the complexity of cellular responses, I hypothesized that the integration of multiple matrix factors as opposed to their individual contribution is key to understanding and controlling cell function. The objective of this thesis was to systematically investigate matrix biochemical and mechanical regulation of multipotent stromal cell (MSC) osteogenesis. First, I demonstrated that substrate stiffness-dependent MSC spreading, proliferation and osteogenic response were differentially regulated by matrix protein type (collagen I vs. fibronectin) and concentration. Second, I developed and characterized a matrix microarray platform that enabled the efficient screening of multiple matrix-derived cues (substrate stiffness, ECM type and density). I implemented the matrix microarray platform together with parametric regression models to elucidate novel matrix interactions in directing mouse MSC osteogenic and adipogenic differentiation. Third, I extended the screening study to examine matrix-dependent human MSC osteogenesis. Non-parametric regression models were used to provide a nuanced description of the multi-factorial matrix regulation in MSC osteogenesis. The response surfaces revealed a biphasic relationship between osteogenesis and substrate stiffness, with the exact location and magnitude of the optimum contingent on matrix composition. Guided by the screening results and perturbation to key cytoskeletal regulators, I identified a novel pathway involving Cdc42 in matrix mechanical and biochemical regulation of MSC osteogenesis. Surprisingly, Cdc42 mediated stiffness-dependent MSC osteogenesis independent of ROCK, suggestive of a contractility-independent mechanism in matrix rigidity signal transduction. In summary, the integration of cell-based arrays and statistical modeling has enabled the systematic investigation of complex cell-matrix interactions. This generalizable approach is readily adaptable to other cellular contexts, complementing hypothesis-driven strategies to facilitate non-intuitive mechanistic discovery. Moreover, the improved understanding of matrix-dependent MSC function also has practical relevance to the development of biomaterials for tissue engineering applications.

Made available in DSpace on 2014-06-11T19:35:10Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-10-08Bitstream added on 2014-06-13T19:45:08Z : No. of bitstreams: 1 limaneto_jf_dr_botfmvz.pdf: 5509939 bytes, checksum: 6d585da226479576f95a5b051bde27a2 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A célula-tronco (CT) é definida como uma célula com capacidade de gerar diferentes tipos celulares e reconstituir diversos tecidos. Além disso, a CT apresenta propriedades de auto-renovação, gerando cópias idênticas a si mesma. De acordo com sua origem, as células-tronco podem ser chamadas de adultas e embrionárias. As células-tronco adultas (CTA) mais utilizadas nas clínicas de terapia celular são as células-tronco hematopoiéticas e as células tronco mesenquimais, encontradas principalmente na medula óssea, tecido adiposo e no sangue do cordão umbilical. As células-tronco embrionárias (CTE) são derivadas da massa celular interna de embriões no estágio de blastocisto. Desta maneira este trabalho teve como objetivo desenvolver uma metodologia adequada para o isolamento, cultivo e caracterização de células tronco embrionárias e mesenquimais de eqüinos, além de verificar a capacidade que as células possuem em se diferenciar in vitro em outros tipos célulares. Foi coletado sangue da medula óssea de eqüinos entre 8 e 15 anos de idade. As células tronco mesenquimais foram isoladas após a primeira e segunda passagem. As células foram caracterizadas com marcadores de superfície CD34 (mononucleares) e CD44 (mesenquimais). Após isolamento e caracterização, as células tronco mesenquimais foram diferenciadas para as linhagens osteogênica, adipogênica, condrogênica e neurogênica. A confirmação da diferenciação das células tronco foi realizada por marcadores teciduais específicos. Estas células também, foram capazes de expressarem marcadores neurais. Para o isolamento das células tronco embrionária eqüina, embriões com oito a nove dias foram coletado e a massa celular interna (MCI) isolada mecanicamente. Após o isolamento, a MCI foi transferida para a placa de cultivo previamente preparada com monocamada de fibroblastos para o desenvolvimento... / The stem cell (SC) is defined as cells with the capacity of generate different cellular types and rebuild various tissues. Moreover, the SC has a selfregenerate ability, generating identical copies of itself. According to its origins, the SC can be named as “adult” or “embryonic”. The adult stem cell (ASC) more often used in clinical trials and cellular therapy, are the hematopoietic stem cells and the mesenchymal stem cells, isolated mainly from the marrow bone, adipose tissue and umbilical cord blood. The embryonic stem cells (ESC) are obtained from the inner cell mass of embryos at the blastocyst stage. In this way the present study had as objective to develop an adequate methodology of isolation, culture and characterization of embryonic and mesechymal stem cells from horses, verifying the capacity of those cells to differentiate in vitro into different cells types. Bone marrow blood was collected from horses, aging from 8 to 15 years and filtered with a donation blood kit filter, to avoid clots. The mesenchymal stem cells were isolated after the first and the second passage. The SC were characterized using surface markers CD34 (monuclear) and CD44 (mesenchymal). After the isolation and characterization, the mesenchymal stem cells were differenced into osteogenic, adipogenic, condrogenic and neurogenic lineage. The cells differentiations were confirmed using specific tissue markers. To isolate the embryonic stem cells equine embryos with 8 to 9 days were used. The inner cell mass (ICM) were extract mechanically and transferred to a culture dish previously prepared with fibroblasts monolayer to colony formation and development. The colonies were characterized with pluripotency markers and then submitted to a differentiation process into neurogenic lineage, confirmed by specific neural tissue markers

A Caderina-E està intimamente relacionada com a transiÃÃo epitelial-mesenquimal e com a progressÃo tumoral em muitos tipos de cÃncer, inclusive no cÃncer colorretal. O objetivo deste trabalho foi avaliar a imunoexpressÃo de Caderina-E no cÃncer colorretal primÃrio e nas respectivas metÃstases linfonodais, na mucosa colÃnica normal, e investigar possÃveis correlaÃÃes desta expressÃo com parÃmetros clÃnicopatolÃgicos. Setenta e sete casos de colectomias por carcinoma colorretal e dez casos de linfonodos metastÃticos, dos arquivos do Departamento de Patologia e Medicina Legal/Universidade Federal do CearÃ, foram utilizados. Realizou-se o Tissue Microarray e imunohistoquÃmica, com anticorpo monoclonal anti-Caderina-E. Foram avaliados os seguintes escores: 0 = ausÃncia de expressÃo; 1 = expressÃo citoplasmÃtica; 2 = expressÃo mista (citoplasmÃtica e membranar); 3 = expressÃo membranar pura. Foi utilizada tanto a classificaÃÃo proposta por Jawhari et al., agrupando os casos em expressÃo anormal (escores 0, 1 e 2) e expressÃo normal (escore 3), como os critÃrios propostos por Almeida et al., agrupando os casos como expressÃo nÃo-membranar (escores 0 e 1) e expressÃo membranar (escores 2 e 3). Os tumores primÃrios tiveram mais casos de expressÃo de Caderina-E anormal em comparaÃÃo com a mucosa normal (p < 0.0001). NÃo houve diferenÃa significante entre expressÃo de Caderina-E no tumor intestinal e em metÃstases linfonodais, embora nestas a expressÃo membranar tenha sido mais freqÃente do que no sÃtio primÃrio. Tumores de cÃlulas agrupadas apresentaram maior expressÃo de Caderina-E membranar do que os de cÃlulas isoladas, tanto utilizando a classificaÃÃo de Jawhari et al. (p = 0.0230), como os critÃrios propostos por Almeida et al. (p = 0.0043). Em conclusÃo, a expressÃo anormal de Caderina-E no tumor primÃrio, com persistÃncia freqÃente da imunomarcaÃÃo membranar associada à marcaÃÃo citoplasmÃtica (marcaÃÃo anormal heterogÃnea ou mista), reforÃa as evidÃncias de que esta alteraÃÃo no cÃncer à mais qualitativa do que propriamente quantitativa. O predomÃnio da expressÃo membranar no sÃtio primÃrio da neoplasia e na metÃstase, com ou sem expressÃo citoplasmÃtica associada, principalmente em tumores de cÃlulas agrupadas, sugere que a presenÃa da Caderina-E à essencial para a invasÃo local e progressÃo tumoral, em oposiÃÃo ao clÃssico paradigma de que a progressÃo tumoral se exacerba com a perda desta molÃcula de adesÃo. / E-cadherin is closely related to epitelial-mesenchymal transition and tumor progression in many cancers, including colorectal cancer. The aim of this study is to evaluate the expression of E-cadherin in primary colorectal cancer as well as in lymph node metastasis, establishing also a comparison with the expression of E-cadherin in normal colonic mucosa. We utilized 77 cases of colectomies for colorectal carcinoma and 10 cases of metastatic lymph nodes from the files of the Department of Pathology and Forensic Medicine/Federal University of Ceara. Tissue microarray and immunohistochemistry were performed with monoclonal anti-E-cadherin, evaluated using the following scores: 0 = no staining; 1 = cytoplasmic staining; 2 = mixed staining (cytoplasmic and membranous); 3 = membranous staining. It was used the classification proposed by Jawahri et al. which includes cases of abnormal expression (0, 1 and 2 scores) and cases of normal expression (3 score), and was also used the classification proposed by Almeida et al. which includes cases of non-membranous expression (0 and 1 scores) and membranous expression (2 and 3 scores). Primary tumors presented more cases of abnormal E-cadherin expression in comparison to normal colonic mucosa (p < 0.0001). There were no differences between E-cadherin expression in the primary tumor in comparison to lymph node metastasis. The grouped cell tumors showed increased expression of E-cadherin in comparison to isolated cell tumors, either using the classification proposed by Jawhari et al. (p = 0.0230) and the classification proposed by Almeida et al. (p = 0.0043). In conclusion, abnormal expression of E-cadherin in the primary tumor, with frequent membranar immunostaining associated with the cytoplasmic marking (abnormal heterogeneous or mixed staining), reinforces the evidence that E-cadherin expression change in cancer is more qualitative than quantitative. The predominance of membranar expression in primary tumor and lymph node metastasis, with or without associated cytoplasmatic expression, particularly in cell-grouped tumors, suggests that E-cadherin presence is essential for local invasion and tumor progression, as opposed to the classical paradigm that tumor progression is exacerbated by the loss of this adhesion molecule.

Abstract How cell and tissue interactions lead to complex structures and differentiated cell types during organogenesis is still one of the most fundermental questions in modern molecular biology. Laminin appears to have a role in branching morphogenesis during organ development. Laminin5 (α3β3γ2) is an epithelium-specific isoform of laminin and previous report has shown that two alternative transcripts for the γ2 chain, the longer γ2 and the shorter γ2*, result from alternative use of the last exon in the human LAMC2 gene. But the transcription of murine laminin γ2 and γ2* and their biological significance have remained unclear. Type XVIII collagen is a newly identified member of the collagen family. It may be involved in the Wnt signaling pathway, since its longest N-terminal variant contains a frizzled domain, which is part of the Wnt receptor and could antagonize Wnt signaling when secreted. Wnt2b is a new member of the Wnt family. Also its function in organogenesis is unknown. In this study, we have investigated the expressions of laminin γ2 and γ2*, type XVIII collagen and Wnt2b during mouse organogenesis. The function of type XVIII collagen in developing lung, kidney and a recombination of ureteric bud and lung mesenchyme tissue and the function of the Wnt-2b gene during kidney organogenesis were studied by using the combined methods of traditional experimental embryology and modern molecular biology. Two alternative laminin γ2 transcripts were demonstrated in mouse. In the developing kidney, the shorter γ2* form was localized in the mesenchyme, whereas the longer γ2 form was only present in the epithelium of the Wolffian duct and in the ureteric bud, indicating different functions for the γ2 variants. Type XVIII collagen was expressed throughout the respective epithelial bud at the initiation of lung and kidney organogenesis. It becomed localized to the epithelial tips in the early-stage lung, while it was confined to the epithelial stalk region and was absent from the nearly formed ureteric tips in the kidney. In recombinants of ureteric bud and lung mesenchyme, the type XVIII collagen expression pattern in the ureteric bud shifted from the kidney to the lung type, accompanied by a shift in epithelial Sonic Hedgehog expression. The lung mesenchyme was also sufficient to induce ectopic lung Surfactant Protein C expression in the ureteric bud. A blocking antibody for the type XVIII collagen reduced the number of epithelial tips in the lung and completely blocked ureteric development with lung mesenchyme, which was associated with a notable reduction in the expression of Wnt2. The shift in type XVIII collagen expression in ureteric bud and lung mesenchyme tissue recombinant was also accompanied by the significant morphological changes in the branching pattern in ureteric bud development. Wnt2b was expressed in numerous developing organs in the mouse embryo, but it was typically localized in the perinephric mesenchymal cells in the region that partly overlaps the presumptive renal stroma at E11.5. Functional studies of the kidney demonstrated that 3T3 cells expressing Wnt2b were not capable of inducing tubule formation but rather stimulated ureteric development. Recombination of ureteric bud treated with cells expressing Wnt2b and isolated kidney mesenchyme resulted in recovery of the expression of epithelial marker genes and better reconstituted organogenesis. Lithium, a known activator of Wnt signaling, was also sufficient to promote ureteric branching in reconstituted kidney in a manner comparable to Wnt2b signaling. Our data suggest that different organ morphogenesis is regulated by an intraorgan patterning process that involves coordination between inductive signals and matrix molecules, such as type XVIII collagen. In the mouse kidney, Wnt2b may act as an early mesenchymal signal controlling morphogenesis of epithelial tissue, and the Wnt pathway may regulate ureteric branching directly.

Abstract The polyamines putrescine, spermidine and spermine are highly regulated polycations present in virtually all cells of higher eukaryotes. They are essential for proper cell growth and differentiation by participating in various physiological processes including DNA, RNA and protein synthesis, apoptosis and interactions with ion-channels. The complexity of polyamine metabolism and the multitude of compensatory mechanisms that are invoked to maintain polyamine homeostasis argue that these molecules are critical for cell survival. The primary aim of this study was to gain a better understanding of the mode of action of polyamines and the regulatory mechanisms in which they are involved. Transgenic mice overexpressing the polyamine biosynthetic enzymes S-AdoMetDC and ODC were found to maintain their polyamine pools by acetylation of spermidine and spermine and an increased export of these acetylated compounds. The expression of various genes was studied as a response to polyamine deprivation in cell- and kidney organ culture. Among these genes acetyl-CoA synthetase and ornithine decarboxylase were demonstrated to be developmentally regulated. Changes in gene expression patterns, with most of the transcripts upregulated in the polyamine-depleted samples, indicated selective stabilization of mRNAs. Polyamines were shown to play an important role in kidney organogenesis as their depletion results in a reduction of ureteric branching and retardation of tubule formation. The selective changes of various genes in the ureteric bud and mesenchyme indicate that polyamines might have a role in the regulation of epithelial-mesenchymal interactions during mouse kidney development.

Glomerular diseases are the leading cause of end stage kidney disease worldwide. Podocyte injury plays a key role in the initiation and development of such diseases, which follow a progressive course due to the limited capacity of podocytes to regenerate. Podocytes are highly specialised, terminally differentiated cells, which play a vital role in the glomerular filtration barrier. They are also the main sites of expression of the Wilms Tumour Suppressor gene, WT1, in the adult. WT1 is a complex gene, which plays an essential role in renal development by controlling the process of mesenchymal to epithelial transition that forms the nephron. Adult podocytes maintain both epithelial and mesenchymal features and continue to express high levels of WT1. Little is known about the role of WT1 in adult podocytes as previous studies have been limited due to the confounding developmental effects and embryonic lethality of existing animal models. This thesis sought to investigate the hypothesis that WT1 is an essential gene in adult kidney and plays a fundamental role in the adult podocyte. Given its role in nephron development, WT1 loss was hypothesised to result in dedifferentiation and an alteration of the epithelial-mesenchymal balance in the podocyte, affecting its specialised function. Using an inducible, conditional animal model of Wt1 loss, Wt1 was deleted from the adult, confirming its essential role in adult kidney. Wt1 deletion resulted in severe podocyte injury and failure of the glomerular filtration barrier, as well as loss of expression of key podocyte genes. Preliminary analysis suggests this was not simply due to podocyte apoptosis and/or detachment, supporting a role for Wt1 in podocyte differentiation. This was corroborated by in vitro studies that demonstrated a requirement for Wt1 for podocyte differentiation. Significantly, Wt1 loss resulted in a marked change in the expression of epithelial and mesenchymal markers in podocytes, with upregulation of mesenchymal characteristics, in keeping with a transitional stage consistent with an earlier developmental form. To investigate the mechanism behind these findings a conditionally immortalised podocyte cell line was generated as a model of Wt1 loss in vitro. In order to confirm and specifically analyse the podocyte phenotype, BAC recombineering was utilised to produce a promoter-reporter transgene construct to attempt to generate a fluorescent-labelled, podocyte specific animal model of Wt1 loss. The findings of this thesis establish that Wt1 is essential for adult podocyte function, and appears to be a key upstream regulator of podocyte differentiation. Extension of this work may allow the identification of potential targets to promote podocyte differentiation and/or regeneration in the setting of acquired and progressive glomerular disease.

Type 1 diabetes mellitus is a metabolic disorder characterized by an autoimmune attack against the insulin producing Beta-cells of the pancreas. Also known as insulin-dependent diabetes, patients must receive exogenous injections of insulin in order to maintain glycemic homeostasis. The necessity of monitoring one’s own blood glucose levels and self-administering insulin is a tedious routine for type 1 diabetics, and this standard treatment option fails to treat any of the underlying causes of the disease. According to van Belle et al, the prevalence of diabetes is rising worldwide amongst all age-groups, from 2.8% in 2000 to an estimated 4.4% by 2030, thus the need to find a more curative treatment approach is eminent. In the emerging field of regenerative medicine, mesenchymal stem cells have been identified as a possible therapeutic tool to replace damaged parenchymal tissue. Along with their ability to modulate the local microenvironment, the introduction of properly differentiated mesenchymal stem cells into patients with Type 1 diabetes may provide a treatment option that helps supplement the lost islet cells without provoking an immune response. Preliminary clinical trials have shown that stem cell therapy decreases the amount of exogenous insulin required daily, decreases fasting glucose levels, decreases amount of glycated hemoglobin and increases C-peptide levels. These four indicators of diabetic control suggest that mesenchymal stem cells are an effective means of helping manage Type 1 diabetes. Still, much research needs to be done to fully understand the biomechanics behind the cells’ actions in order to expand human clinical trials. Although complete insulin independence is rarely achieved in patients receiving mesenchymal stem cell treatment, the promising results shown so far suggest more studies be undertaken in hopes of finding a corrective approach to treat Type 1 diabetes.

BACKGROUND: Human mesenchymal progenitor cells are multipotent cells that can be harvested from various adult and fetal tissues. They exhibit the potential to differentiate into several cell lineages, most notably osteogenic, chondrogenic, and adipogenic lineages. Conditions such as osteoporosis, metabolic disease, and arthritis are examples of dysfunction of tissues formed by the mesenchyme. The inability of these conditions to be healed by the body’s own mechanisms has raised considerable interest in the potential of using mesenchymal progenitor cells as a therapeutic intervention. This concept opens the possibility of harvesting mesenchymal progenitor cells from an individual, growing them into the desired tissue, and implanting them back into the individual. Treatment of this nature is much less invasive than current methods, overcomes rejection by the immune system, and could potentially demonstrate better outcomes in individuals suffering from degenerative disease of the mesenchyme. AIMS/OBJECTIVES: The aims of this study were to determine and to characterize the differentiation of human adipose-derived mesenchymal progenitor cells into osteocytes, chondrocytes, and adipocytes. The differentiation capacity of the mesenchymal progenitor cells was evaluated through cell staining, immunofluorescence, and RNA sequencing. METHODS: Subcutaneous adipose tissue was collected from patients undergoing elective panniculectomies. The abdominal panniculus was liposuctioned, and small explants of fat were embedded in Matrigel. Mesenchymal progenitor cells were extracted from the explants and plated for differentiation into osteogenic, chondrogenic, and adipogenic lineages. Control cells were grown in parallel in basal media to confirm differentiation. Dye staining for differentiation was performed with Alizarin Red S, Alcian Blue, and Oil Red O, and immunofluorescence staining was performed to indicate lineage-specific markers for differentiation. RNA sequencing was also completed on the different cell lineages. RESULTS: Human adipose-derived mesenchymal progenitor cells displayed the capacity to differentiate into osteogenic, chondrogenic, and adipogenic lineages as evidenced by dye staining. Osteogenic differentiation was confirmed with Alizarin Red S staining of calcium deposits in the differentiated cells, whereas staining in the control resulted in no calcium deposits. Alcian Blue staining confirmed chondrogenic differentiation as glycoproteins secreted by the differentiated cells were evident and different in morphology compared with the control cells. Oil Red O staining indicated adipogenic differentiation by showing lipid droplets in the differentiated cells and no lipid droplets in the control. RNA sequencing provided support that lineage differentiation was successful. Immunofluorescence staining further proved that differentiated cells expressed lineage-specific proteins and demonstrated morphological differences. CONCLUSIONS: This study demonstrates that mesenchymal progenitor cells harvested from human adipose tissue have the potential to differentiate into adipogenic, chondrogenic, and osteogenic cell lineages when induced with differentiation media. The differentiation of these cells can be assessed with dye staining, RNA sequencing, and immunofluorescence staining methods. Further studies should be done to investigate the potential of mesenchymal progenitor cells for therapeutic interventions in the treatment of various illnesses related to the mesenchyme.

No description available.

Introduction: MSCs are multipotent progenitor cells that can differentiate into various cell lineages, such as osteoblasts, chondrocytes and adipocytes. MSCs synthesize abundant secretion factors to extracellular matrix which contain a variety of growth factors, cytokines and microRNAs. Secretion factors could stimulate the regeneration and differentiation of surrounding cells, but their underlying mechanism still remains elusive. We hypothesized that secretion factors from different tissues derived MSCs had potential to promote MSCs differentiation and musculoskeletal tissue regeneration. We also suggested that microRNAs played an essential role in the effects of secretion factors. In present study, we investigated the effects of secretion factors obtained from different tissues derived MSCs (umbilical cord, dental pulp, gingiva and adipose tissue) on multi-differentiation potentials of MSCs, including osteogenesis, chondrogenesis, tenogenesis, neurogenesis and adipogenesis. Moreover, we illustrated the effects of umbilical cord derived MSC (UCMSC) secretion on bone, cartilage and tendon tissue repair. We further revealed that microRNAs may impact the effect of secretion factors on MSCs osteogenic differentiation. / Methods: Human bone marrow MSCs (hBMSCs) were incubated with various differentiation induction media. Secretion factors were used as supplement. Different animal models of tissue repair (bone, cartilage and tendon) were employed for study of the effects of secretion factors on tissue healing. miRNA microarray was performed to find the potential effective miRNAs in secretion factors. Real time qRT-PCR, microCT, mechanical test, immunohistological analysis and various staining methods were employed as outcome measurements. / Results: We found that both UCMSC and dental pulp derived MCS secretion could initiate osteogenic differentiation of hBMSCs without osteogenic induction medium. UCMSC secretion had positive effect on chondrogenic and tenogenic differentiation of MSCs and inhibitory effect on adipogenesis of hBMSCs. Our results showed that UCMSC secretion in HA/TCP scaffolds with hBMSCs promoted ectopic bone formation in nude mice. UCMSC secretion with rat BMSCs in hyaluronic hydrogel significantly enhanced the bone repair of rat calvarial bone critical defect. To reveal the underlying mechanism, secretion factors were analyzed by miRNA microarray. Among the differentially expressed microRNAs, we found miR-1237 could promote osteogenesis while miR-3676 could inhibit osteogenic differentiation of MSCs. / Conclusions: This study indicated that among secretion factors from MSCs form four types tissues, UCMSC secretion could initiate osteogenesis of MSCs and promote bone repair. We also demonstrated that microRNAs from secretion had impact on osteogenic differentiation of MSCs. Our study showed clinical potential of UCMSC secretion in bone regeneration, and more research are needed for optimizing the preparation and delivery of the MSCs secretive factors, as well as to understand their mechanisms of action. / 前言：間充質幹細胞是具有強大分化潛能的始祖細胞。間充質幹細胞可以分化為多種細胞系，例如成骨細胞，軟骨細胞和脂肪細胞。間充質幹細胞合成并釋放大量分泌素到細胞外基質中。這些分泌素包括多種生長因子，細胞因子和微小核糖核酸。分泌素能夠刺激周圍細胞的再生和分化，但是分泌素的作用機理還不是很清楚。我們認為，不同組織來源的間充質幹細胞分泌素有可能會促進間充質幹細胞的多系分化和骨骼肌肉組織的再生，並且微小核糖核酸在分泌素的效應中發揮了重要作用。我們首先研究了臍帶，牙髓，牙齦和脂肪來源的間充質幹細胞分泌素對于間充質幹細胞的分化能力的作用。我們還對臍帶幹細胞分泌素在骨，軟骨和肌腱修復的效果做了進一步的研究。另外，我們還發現分泌素中的微小核糖核酸在間充質幹細胞的成骨分化方面有一定的效果。 / 方法：我們用人間充質幹細胞來進行誘導分化實驗。臍帶，牙髓，牙齦和脂肪來源的間充質幹細胞的分泌素用於細胞培養基的補充。在體內實驗中我們用了不同的動物模型，把填充物和分泌素一起種植在動物體內。我們利用微小核糖核酸陣列技術來檢測分泌素中的有效微小核糖核酸。我們使用了定量聚合酶鏈反應技術，微型計算機斷層掃描成像，力學測試，免疫組織分析和多種染色方法。 / 結果：我們發現臍帶和牙髓間充質幹細胞分泌素可以在沒有成骨誘導培養基的情況下啟動骨髓間充質幹細胞的成骨分化。臍帶間充質幹細胞對成軟骨和成肌腱分化起到積極作用，而且可以抑制脂肪分化。我們發現在羥基磷灰石/磷酸三鈣材料中，臍帶間充質幹細胞分泌素與人骨髓間充質幹細胞可以共同促進裸鼠的異位成骨。臍帶間充質幹細胞分泌素與鼠骨髓間充質幹細胞一起用於透明質酸水凝膠中能夠加快大鼠頭骨缺損的修復。為了揭示分泌素的作用機理，我們用微小核糖核酸陣列技術來檢測分泌素。在表達不同的微小核糖核酸之中，我們發現miR-1237可以促進間骨髓間充質幹細胞的成骨分化，而miR-3676能夠抑制骨髓間充質幹細胞成骨分化。 / 結論：本研究表明，在四種不同來源的分泌素中，臍帶間充質幹細胞分泌素可以啟動骨髓間充質幹細胞的成骨分化，同時加快骨組織修復。我們發現微小核糖核酸在分泌素的促進間骨髓間充質幹細胞成骨分化的效果中發揮了一定的作用。我們的研究表明，使用臍帶間充質幹細胞分泌素修復骨組織具有廣泛的臨床應用前景。間充質幹細胞分泌素的生產，使用過程和作用機理還有待于進一步的優化和研究。 / Wang, Kuixing. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 131-147). / Abstracts also in Chinese. / Title from PDF title page (viewed on 01, November, 2016). / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.