本文選題：大鼠骨髓間充質(zhì)干細(xì)胞 + 端粒酶　； 參考：《吉林大學(xué)》2009年博士論文

【摘要】： 本研究以大鼠骨髓間充質(zhì)干細(xì)胞(rat mesenchymal stem cells, rMSCs)為對(duì)象,觀察體外傳代培養(yǎng)的rMSCs衰老的發(fā)生,并探討端粒酶在該過(guò)程中的作用及酶活性調(diào)節(jié)相關(guān)的信號(hào)傳導(dǎo)通路。 本實(shí)驗(yàn)聯(lián)合應(yīng)用密度梯度離心、貼壁篩選和單克隆培養(yǎng)法,在體外快速分離獲得了高純度的rMSCs,并能進(jìn)行傳代培養(yǎng)。但隨著傳代培養(yǎng)時(shí)間的延長(zhǎng),rMSCs的生物學(xué)特性發(fā)生改變,逐漸呈現(xiàn)衰老的特征,主要表現(xiàn)為:細(xì)胞體積增大、粗面內(nèi)質(zhì)網(wǎng)擴(kuò)張、次級(jí)溶酶體出現(xiàn)、細(xì)胞器空泡化、核固縮等形態(tài)改變;同時(shí)細(xì)胞的生長(zhǎng)增殖能力以及多向分化潛能均降低。此外,在rMSCs衰老發(fā)生過(guò)程中細(xì)胞的端粒酶活性逐漸降低,端粒長(zhǎng)度也隨之縮短。進(jìn)一步,本實(shí)驗(yàn)應(yīng)用RNAi技術(shù)沉默處于生長(zhǎng)旺盛狀態(tài)rMSCs的端粒酶活性,發(fā)現(xiàn)能促進(jìn)細(xì)胞體積增大、細(xì)胞器空泡化等衰老形態(tài)特征的出現(xiàn),減弱其生長(zhǎng)增殖能力,促進(jìn)細(xì)胞凋亡發(fā)生率,即沉默端粒酶活性能夠誘導(dǎo)rMSCs發(fā)生衰老;而在衰老rMSCs中過(guò)表達(dá)端粒酶活性,則能增強(qiáng)細(xì)胞的生長(zhǎng)增殖能力,降低細(xì)胞凋亡發(fā)生率,說(shuō)明端粒酶活性的改變能夠一定程度調(diào)控rMSCs衰老的發(fā)生和進(jìn)程。此外,本實(shí)驗(yàn)還觀察到分別沉默和過(guò)表達(dá)端粒酶活性可以減弱和增強(qiáng)rMSCs中PI3K/AKT信號(hào)傳導(dǎo)通路下游靶蛋白AKT的磷酸化水平,而特異性抑制劑LY294002抑制PI3K/AKT信號(hào)傳導(dǎo)通路后,rMSCs的端粒酶活性也隨之減弱,端粒長(zhǎng)度隨之縮短,提示端粒酶在rMSCs衰老發(fā)生中的作用是通過(guò)或是受PI3K/AKT信號(hào)傳導(dǎo)通路調(diào)控。 綜上所述,本實(shí)驗(yàn)觀察了體外傳代培養(yǎng)的rMSCs衰老的發(fā)生過(guò)程,并從正、反兩方面探討了端粒酶在此過(guò)程中的作用及酶活性調(diào)節(jié)相關(guān)的信號(hào)傳導(dǎo)通路,為更好的了解rMSCs衰老的發(fā)生提供了新的實(shí)驗(yàn)基礎(chǔ),期望能為端粒酶在抗衰老的應(yīng)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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類號(hào)】：R346

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4 周s，

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