【作者】 杨旭芳；

【导师】 李玉林； 何旭；

【作者基本信息】 吉林大学 ， 病理与病理生理学， 2010， 博士

【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﹢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。更多还原

【Abstract】 With the increasing incidence of vascular diseases, the desire for the vascular transplantation materials is also continually expanded. In order to solve a variety of problems about the synthetic materials and the autologous vascular transplantation, scientists tried many ways to construct tissue-engineered blood vessels. And how to obtain the‘seeding cells’is one of the challenges for us.Up to now,some achievements has been got in the treatment of ischemic diseases with endothelial cells. Considering the difficulty in obtaining and amplifying endothelial cells,seeking for suitable seeding cells to construct tissue-engineered blood vessels has been a hot pot. The stem cells have been the favorite choice for seeding cells in tissue engineering due to highly proliferative ability and multipotentiality.Nowadays, there are two kinds of stem cells: embryonic stem cells and adult stem cells. Due to the limitation of embryonic stem cells,such as ethical problems and inconvenient harvest, adult stem cells have gradually become investigative focus. Compared with bone marrow-derived mesenchymal stem cells, ADSCs(Human adipose-derived stem cells) are also multipotent stem cells derived from the mesoderm, which possess potent proliferative capacity and self-renewal and multilineage differentiation.But ADSCs have some merit, including drawing the materials easily, slightly injury, multiple amplification in vitro and induced differentiation easily, which make them become the main source of tissue engineered vascular seeding cells.In 2001, Zuk[27]obtained adipose-derived stem cells from human adipose tissue. Subsequently scientists modified several methods. So far,there is no acceptable, simple or efficient method for isolating and cultivating highly homogenous and undifferentiated hADSCs. Moreover, methods for identification have not been fully established. Therefore, it is inevitable to establish highly efficient method to isolate and identify hADSCs.The molecular mechanism of hADSCs directional differentation into endothelial cells is not clear. Only the mechanism of differentiated regulation are found out, it will offer abundant seeding cells for tissue engineering. Because both the stem cell research and proteomics research are hot spots in life sciences in the 21th century, if we want to recognize the complex activities of stem cells, we must focus on the proteins from the whole and dynamic point of view.In the present study, hADSCs were isolated, cultivated and amplified,and the biological characteristics of hADSCs were identified as well. Then hADSCs were induced into endothelial cells by utilizing cytokine and extracellular matrix. Furthermore, hADSCs differential proteins in directional differentiation into endothelial cells were screened by proteomics techniques.The results show that we have successfully isolated and cultured highly homogenous and undifferentiated hADSCs, which have multilineage differentiation properties. The most important finding is that one gram of adipose tissue can yield～1×10~6 adipose-derived mesenchymal stem cells, equivalent to the yield from 40ml bone marrow. In addition,hADSCs are negative for histocompatibility complex molecules (HLA-DR), which implies that hADSCs are suitable for allograft without MHC limitation. hADSCs we have got can be passaged, freezed and revived stably. These characteristics will better meet the demands of cell transplant and tissue engineering.The multi-differentiation ability is the most important identification for stem cells[20]. The results show that human adipose-derived mesenchymal stem cells has potentiality to be induced into adipocytes, osteocytes and endothelial cells. In this experiment, we compared the differentiation process at different time points. With the extension of time, the capacity of being induced into adipocytes,or osteocytes is also increased.We study the factors which impact hADSCs directional differentiation into endothelial from the cytokine and extracellular matrix point of view,and find that the combination of FN as extracellular matrix and endothelial cell supporting culture medium EGM2-MV as well as high concentration VEGF165(50ng/ml)is optimal for endothelial differentiation.We screened directional differentially expressed proteins of in hADSCs differentiation into endothelial cells by using proteomic techniques, then we identifed these proteins by using MALDITOF -TOF, and we got 10 key regulatory proteins related to the differentiation of hADSCs into endothelial cells, which provides an important experimental foundation for understanding the differentiation process in the regulatory mechanism.In conclusion, we have successfully isolated and cultured highly homogenous and undifferentiated hADSCs, optimized the induced differentiation system of endothelial cells, and for the first time in this experiment, we screened directional differentially expressed proteins in hADSCs differentiation into endothelial cells by proteomic technologies. The results provides not only a theoretical basis for the molecular mechanism of hADSCs induced differentiation into endothelial cells, but also a sufficient experimental evidence in using hADSCs to treat ischemic diseases or vascular-dependent diseases and promote blood vessel regeneration in tissue-engineered organs.更多还原