人脂肪干细胞的获取及其定向分化为内皮细胞差异蛋白筛选的实验研究

发布时间：2018-02-13 15:19

本文关键词： 人脂肪干细胞分离鉴定内皮分化蛋白组学差异蛋白　出处：《吉林大学》2010年博士论文　论文类型：学位论文

【摘要】： 为解决血管组织工程种子细胞来源不足问题,及探索干细胞定向内皮分化的分子机制,本实验以人脂肪干细胞(hADSCs)为研究对象,进行了hADSCs体外分离、培养、扩增和生物学特性的鉴定,最佳内皮细胞诱导分化体系的探索及应用蛋白质组学技术从蛋白质角度筛选hADSCs内皮分化过程中所表达的差异蛋白。应用胶原酶消化和贴壁筛选联合培养的方法,从人脂肪组织中快速分离获得了hADSCs,经一般生物学特性的检测,证实了我们获得的hADSCs具有较高的纯度,能够稳定的进行传代培养以及冻存、复苏,这符合组织工程对种子细胞的要求。从细胞因子与细胞外基质的角度探讨其对hADSCs定向内皮分化的影响,最终摸索出以纤维粘连蛋白(FN)作为细胞外基质与富含多种生长因子的内皮细胞支持液EGM2-MV㧏高浓度VEGF165(50ng/ml)组成的诱导条件,构成了hADSCs最佳内皮细胞诱导分化的体系。应用蛋白质组学技术首次从蛋白质角度筛选出了hADSCs定向分化为内皮细胞的差异蛋白。为进一步了解其分化过程中的调控机制提供了重要的实验基础。综上所述,本研究建立了hADSCs体外分离培养及大规模扩增的简易技术;建立了hADSCs体外最佳的内皮细胞诱导分化体系;筛选出hADSCs内皮分化过程中的差异蛋白,初步分析发现了内皮分化相关的10个重要蛋白。上述研究结果为探索hADSCs定向分化的分子机制提供了理论基础,也为人们更好地将hADSCs应用于临床治疗缺血性疾病或血管依赖性疾病,促进组织工程化器官的血管再生,提供了充分的实验依据。
[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.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R329.29

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