本文關鍵詞： 人脂肪干細胞 分離 鑒定 內(nèi)皮分化 蛋白組學 差異蛋白　出處：《吉林大學》2010年博士論文　論文類型：學位論文

【摘要】： 為解決血管組織工程種子細胞來源不足問題,及探索干細胞定向內(nèi)皮分化的分子機制,本實驗以人脂肪干細胞(hADSCs)為研究對象,進行了hADSCs體外分離、培養(yǎng)、擴增和生物學特性的鑒定,最佳內(nèi)皮細胞誘導分化體系的探索及應用蛋白質(zhì)組學技術從蛋白質(zhì)角度篩選hADSCs內(nèi)皮分化過程中所表達的差異蛋白。 應用膠原酶消化和貼壁篩選聯(lián)合培養(yǎng)的方法,從人脂肪組織中快速分離獲得了hADSCs,經(jīng)一般生物學特性的檢測,證實了我們獲得的hADSCs具有較高的純度,能夠穩(wěn)定的進行傳代培養(yǎng)以及凍存、復蘇,這符合組織工程對種子細胞的要求。從細胞因子與細胞外基質(zhì)的角度探討其對hADSCs定向內(nèi)皮分化的影響,最終摸索出以纖維粘連蛋白(FN)作為細胞外基質(zhì)與富含多種生長因子的內(nèi)皮細胞支持液EGM2-MV㧏高濃度VEGF165(50ng/ml)組成的誘導條件,構成了hADSCs最佳內(nèi)皮細胞誘導分化的體系。應用蛋白質(zhì)組學技術首次從蛋白質(zhì)角度篩選出了hADSCs定向分化為內(nèi)皮細胞的差異蛋白。為進一步了解其分化過程中的調(diào)控機制提供了重要的實驗基礎。 綜上所述,本研究建立了hADSCs體外分離培養(yǎng)及大規(guī)模擴增的簡易技術;建立了hADSCs體外最佳的內(nèi)皮細胞誘導分化體系;篩選出hADSCs內(nèi)皮分化過程中的差異蛋白,初步分析發(fā)現(xiàn)了內(nèi)皮分化相關的10個重要蛋白。上述研究結果為探索hADSCs定向分化的分子機制提供了理論基礎,也為人們更好地將hADSCs應用于臨床治療缺血性疾病或血管依賴性疾病,促進組織工程化器官的血管再生,提供了充分的實驗依據(jù)。
[Abstract]:In order to solve the problem of the shortage of seed cells in vascular tissue engineering and to explore the molecular mechanism of endothelial differentiation of stem cells, human adipose stem cells (hADSCs) were isolated and cultured in vitro from human adipose stem cells (hADSCs). Amplification and identification of biological characteristics, exploration of the best system of endothelial cell differentiation and screening of differential proteins expressed in hADSCs endothelial differentiation from the point of view of protein by proteomics. By means of collagenase digestion and co-culture with adherent screening, hADSCs were rapidly isolated from human adipose tissue. The detection of general biological characteristics showed that the obtained hADSCs had high purity. It can be subcultured stably, frozen and resuscitated, which accords with the requirement of tissue engineering for seed cells. The effects of cytokines and extracellular matrix on the differentiation of hADSCs oriented endothelial cells were studied. Finally, using fibronectin (FN) as extracellular matrix and endothelial cell supporting fluid (EGM2-MV) rich in multiple growth factors, EGM2-MV? The inductive conditions of high concentration VEGF165 ~ (50 ng / ml), In order to understand the regulation of the differentiation process of hADSCs, the differential proteins that differentiate hADSCs into endothelial cells were screened out from the point of view of protein by proteomics for the first time. The mechanism provides an important experimental basis. To sum up, we established a simple technique of isolation, culture and large-scale amplification of hADSCs in vitro, established the best endothelial cell differentiation system in vitro for hADSCs, and screened out the differential proteins in the process of hADSCs endothelial differentiation. Ten important proteins related to endothelial differentiation were found by preliminary analysis. These results provide a theoretical basis for exploring the molecular mechanism of hADSCs directional differentiation. It also provides a sufficient experimental basis for the clinical application of hADSCs in the treatment of ischemic or vascular dependent diseases and the promotion of vascular regeneration in tissue engineering organs.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2010
【分類號】：R329.29

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本文編號：1508468