Slingshot-1L磷酸酶在人骨髓间充质干细胞成心肌诱导分化中的作用研究

发布时间：2018-03-05 08:29

  本文选题：人骨髓间充质干细胞　切入点：成心肌分化　出处：《吉林大学》2010年博士论文　论文类型：学位论文

【摘要】： 本研究以体外培养的hMSCs为对象,联合应用密度梯度离心法,贴壁筛选法和单克隆培养法分离培养出高纯度的hMSCs,在体外可以传代培养和大量扩增,细胞在12代前生物学特性稳定,仍保持未分化状态,可作为后续实验的细胞来源。 应用5-Aza对hMSCs进行诱导,分别从形态特征、超微结构、mRNA水平、蛋白质水平以及电生理特性上对诱导后的hMSCs进行联合鉴定,证明诱导后的细胞已经成功向心肌细胞分化,即成功建立了体外hMSCs成心肌诱导体系。 经检测,5-Aza诱导后的hMSCs向心肌细胞分化的过程中Slingshot-1L(SSH-1L)的表达量呈一定的上升趋势,而SSH特异性底物Cofilin的磷酸化水平在诱导后呈明显的下降趋势,提示在hMSCs向心肌细胞分化的过程中SSH-1L可能被激活且对调控hMSCs向心肌细胞分化具有重要的作用。 本研究应用靶向SSH-1L基因的逆转录病毒表达载体pLNCX-SSH-1L转染至hMSCs,成功建立了过表达SSH-1L基因的hMSCs稳定转染细胞系,并研究SSH-1L在hMSCs成心肌诱导分化中的作用。结果显示,与对照组相比,SSH-1L基因转染组的hMSCs向心肌细胞的分化能力明显增强,包括从细胞形态,微丝骨架和电生理特性上加速hMSCs向心肌细胞的变化,上调心肌相关基因及心肌相关蛋白的表达水平。说明SSH-1L对于hMSCs向心肌细胞的分化具有明显的促进作用。 综上所述,本研究从不同角度深入地探讨了SSH-1L在骨髓间充质干细胞成心肌诱导分化中的作用。为进一步明确hMSCs向心肌细胞分化的机制提供有力的实验依据,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.
【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

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