本文選題：人骨髓間充質(zhì)干細(xì)胞　切入點(diǎn)：成心肌分化　出處：《吉林大學(xué)》2010年博士論文　論文類型：學(xué)位論文

【摘要】： 本研究以體外培養(yǎng)的hMSCs為對(duì)象,聯(lián)合應(yīng)用密度梯度離心法,貼壁篩選法和單克隆培養(yǎng)法分離培養(yǎng)出高純度的hMSCs,在體外可以傳代培養(yǎng)和大量擴(kuò)增,細(xì)胞在12代前生物學(xué)特性穩(wěn)定,仍保持未分化狀態(tài),可作為后續(xù)實(shí)驗(yàn)的細(xì)胞來(lái)源。 應(yīng)用5-Aza對(duì)hMSCs進(jìn)行誘導(dǎo),分別從形態(tài)特征、超微結(jié)構(gòu)、mRNA水平、蛋白質(zhì)水平以及電生理特性上對(duì)誘導(dǎo)后的hMSCs進(jìn)行聯(lián)合鑒定,證明誘導(dǎo)后的細(xì)胞已經(jīng)成功向心肌細(xì)胞分化,即成功建立了體外hMSCs成心肌誘導(dǎo)體系。 經(jīng)檢測(cè),5-Aza誘導(dǎo)后的hMSCs向心肌細(xì)胞分化的過(guò)程中Slingshot-1L(SSH-1L)的表達(dá)量呈一定的上升趨勢(shì),而SSH特異性底物Cofilin的磷酸化水平在誘導(dǎo)后呈明顯的下降趨勢(shì),提示在hMSCs向心肌細(xì)胞分化的過(guò)程中SSH-1L可能被激活且對(duì)調(diào)控hMSCs向心肌細(xì)胞分化具有重要的作用。 本研究應(yīng)用靶向SSH-1L基因的逆轉(zhuǎn)錄病毒表達(dá)載體pLNCX-SSH-1L轉(zhuǎn)染至hMSCs,成功建立了過(guò)表達(dá)SSH-1L基因的hMSCs穩(wěn)定轉(zhuǎn)染細(xì)胞系,并研究SSH-1L在hMSCs成心肌誘導(dǎo)分化中的作用。結(jié)果顯示,與對(duì)照組相比,SSH-1L基因轉(zhuǎn)染組的hMSCs向心肌細(xì)胞的分化能力明顯增強(qiáng),包括從細(xì)胞形態(tài),微絲骨架和電生理特性上加速hMSCs向心肌細(xì)胞的變化,上調(diào)心肌相關(guān)基因及心肌相關(guān)蛋白的表達(dá)水平。說(shuō)明SSH-1L對(duì)于hMSCs向心肌細(xì)胞的分化具有明顯的促進(jìn)作用。 綜上所述,本研究從不同角度深入地探討了SSH-1L在骨髓間充質(zhì)干細(xì)胞成心肌誘導(dǎo)分化中的作用。為進(jìn)一步明確hMSCs向心肌細(xì)胞分化的機(jī)制提供有力的實(shí)驗(yàn)依據(jù),指明了新的研究方向。
[Abstract]:In this study, high purity hMSCs were isolated from hMSCs cultured in vitro by density gradient centrifugation, adherent screening and monoclonal culture. The cells were subcultured and expanded in vitro, and the biological characteristics of the cells were stable before 12 passages. It remains undifferentiated and can be used as a cell source for subsequent experiments. HMSCs was induced by 5-Aza, and the induced hMSCs was identified from morphological characteristics, ultrastructural mRNA level, protein level and electrophysiological characteristics, respectively. The results showed that the induced cells had been successfully differentiated into cardiomyocytes. The myocardial induction system of hMSCs in vitro was established successfully. During the differentiation of hMSCs into cardiomyocytes induced by 5-Aza, the expression of Slingshot-1LnSSH-1L) increased to a certain extent, while the phosphorylation level of Cofilin, the specific substrate of SSH, decreased significantly after induction. It is suggested that SSH-1L may be activated in the process of hMSCs differentiation into cardiomyocytes and may play an important role in regulating the differentiation of hMSCs into cardiomyocytes. In this study, a stable transfection cell line of hMSCs expressing SSH-1L gene was successfully established by transfection into hMSCs with retrovirus expression vector pLNCX-SSH-1L targeting SSH-1L gene, and the role of SSH-1L in myocardial differentiation induced by hMSCs was studied. Compared with the control group, the differentiation ability of hMSCs into cardiomyocytes was significantly enhanced in SSH-1L gene transfection group, including the acceleration of hMSCs to cardiomyocytes from cell morphology, microfilament skeleton and electrophysiological characteristics. The expression of myocardial related genes and myocardial related proteins was up-regulated, which indicated that SSH-1L could promote the differentiation of hMSCs into cardiomyocytes. In conclusion, this study explored the role of SSH-1L in myocardial differentiation of bone marrow mesenchymal stem cells from different angles, and provided a powerful experimental basis for further clarifying the mechanism of hMSCs differentiation into cardiomyocytes. The new research direction is pointed out.
【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】：2010
【分類號(hào)】：R329

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本文編號(hào)：1569485