体外培养rMSCs衰老的发生与端粒酶及其相关信号传导通路的研究

发布时间：2018-07-01 15:29

本文选题：大鼠骨髓间充质干细胞 + 端粒酶　；参考：《吉林大学》2009年博士论文

【摘要】： 本研究以大鼠骨髓间充质干细胞(rat mesenchymal stem cells, rMSCs)为对象,观察体外传代培养的rMSCs衰老的发生,并探讨端粒酶在该过程中的作用及酶活性调节相关的信号传导通路。本实验联合应用密度梯度离心、贴壁筛选和单克隆培养法,在体外快速分离获得了高纯度的rMSCs,并能进行传代培养。但随着传代培养时间的延长,rMSCs的生物学特性发生改变,逐渐呈现衰老的特征,主要表现为:细胞体积增大、粗面内质网扩张、次级溶酶体出现、细胞器空泡化、核固缩等形态改变;同时细胞的生长增殖能力以及多向分化潜能均降低。此外,在rMSCs衰老发生过程中细胞的端粒酶活性逐渐降低,端粒长度也随之缩短。进一步,本实验应用RNAi技术沉默处于生长旺盛状态rMSCs的端粒酶活性,发现能促进细胞体积增大、细胞器空泡化等衰老形态特征的出现,减弱其生长增殖能力,促进细胞凋亡发生率,即沉默端粒酶活性能够诱导rMSCs发生衰老;而在衰老rMSCs中过表达端粒酶活性,则能增强细胞的生长增殖能力,降低细胞凋亡发生率,说明端粒酶活性的改变能够一定程度调控rMSCs衰老的发生和进程。此外,本实验还观察到分别沉默和过表达端粒酶活性可以减弱和增强rMSCs中PI3K/AKT信号传导通路下游靶蛋白AKT的磷酸化水平,而特异性抑制剂LY294002抑制PI3K/AKT信号传导通路后,rMSCs的端粒酶活性也随之减弱,端粒长度随之缩短,提示端粒酶在rMSCs衰老发生中的作用是通过或是受PI3K/AKT信号传导通路调控。综上所述,本实验观察了体外传代培养的rMSCs衰老的发生过程,并从正、反两方面探讨了端粒酶在此过程中的作用及酶活性调节相关的信号传导通路,为更好的了解rMSCs衰老的发生提供了新的实验基础,期望能为端粒酶在抗衰老的应用研究提供新的思路。
[Abstract]:In this study, the aging of rat bone marrow mesenchymal stem cells (rat mesenchymal stem cells, rMSCs) in vitro was observed, and the role of telomerase in the process and signal transduction pathway related to the regulation of enzyme activity were investigated. High purity rMSCs was obtained by density gradient centrifugation, adherent screening and monoclonal culture in vitro. However, the biological characteristics of rMSCs were changed with the prolongation of passage time, and gradually showed the characteristics of senescence, such as the enlargement of cell volume, the dilatation of rough endoplasmic reticulum, the appearance of secondary lysosome and the vacuolation of organelle. At the same time, the ability of cell growth and proliferation and the potential of multidirectional differentiation were decreased. In addition, telomerase activity and telomere length of rMSCs decreased gradually during aging. Furthermore, the RNAi technique was used to silence telomerase activity of rMSCs in the growing state. It was found that RNAi could promote the appearance of aging morphological characteristics such as cell volume, cell organ vacuolation, and weaken the ability of growth and proliferation of rMSCs. Promoting apoptosis rate, that is, silencing telomerase activity can induce senescence of rMSCs, but over-expression of telomerase activity in aging rMSCs can enhance cell growth and proliferation ability and decrease apoptosis rate. The changes of telomerase activity can regulate the occurrence and process of rMSCs aging to some extent. In addition, the silencing and overexpression of telomerase activity could attenuate and enhance the phosphorylation of the downstream target protein AKT of PI3K / AKT signal transduction pathway in rMSCs. The specific inhibitor LY294002 inhibited the telomerase activity of rMSCs and shortened the telomere length after inhibiting the PI3K / AKT signal transduction pathway, suggesting that the role of telomerase in the aging of rMSCs was regulated by or by the PI3K / AKT signal transduction pathway. To sum up, the aging process of rMSCs cultured in vitro was observed, and the role of telomerase in the process and the signal transduction pathway related to the regulation of enzyme activity were discussed from both positive and negative aspects. It provides a new experimental basis for better understanding of the occurrence of rMSCs, and it is expected to provide new ideas for the application of telomerase in the research of anti-aging.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R346

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