【作者】 张慕蕊；

【导师】 李玉林；

【作者基本信息】 吉林大学 ， 病理学与病理生理学， 2010， 博士

【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。更多还原

【Abstract】 Adult cardiomyocytes(CMs) belong to permanent cells with limited ability of regeneration. Therefore, people are trying to seach newer ways to make the infracted cardiomyocytes to regenerate and promote revascularization.Recently, stem cellular transplantation has the possibility to replace infarctial cardiomyocytes and to restore function. So it becomes one of the hottest reach fields in rencent years.Mesenchymal stem cells(MSCs)are the stem cells derived from mesoderm, possessing great multi-potent differentiation ability. MSCs can differentiate into many different kinds of cells under different environments.But the mechanisms of MSCs differentiated into cardiomyocytes are still not very clear . As reported, cytoskeleton can play an important role in the process of MSCs differentiation.Slingshot(SSH) is a specific cofilin phosphatase. It can activate cofilin, inhibit the polymerization of actin filament,as a result, it participates in the rearrangement of cytoskeleton. This indicate that SSH may educe important effect in the process of MSCs’differentiation. Therefore, the stable transfected hMSCs cell line was established to study the effect of SSH phosphatase on hMSCs differentiation into cardiomyocytes. It may provide helpful theory support and laboratory basis for further study. A series of experiments were designed to investigate the effect of SSH-1Lphosphatase on hMSCs differentiation into cardiomyocytes.Reslults:1、A standardized platform has been established for hMSCs and the hMSCs maintaining stable characteristics, providing an enough cell source for further study.2、We determined the proper inducing concentration of 5-Aza is 10μM by cell proliferation detection and observed the morphology of hMSCs differentiate into CMs.3、We hint that 5-Aza can successfully induce hMSCs differentiating into CMs.4、The retroviral vector pLNCX-SSH-1L was transfected into packaging cell PA317 by lipofectamine. The transfectants were selected and viral titers were 5×108 CFU /L,collect viral supernatant to infect hMSCs, abtain the monoclones overexpressing SSH-1L. 5、We carryed out a series of detections by Immunocytochemical staining, immuno- fluorescence, RT-PCR and Western blot to confirm that the stable transfected hMSCs cell line over-expression Slingshot mediated by retrovirus was established successfully, providing an enough cell source for further study on the effect of SSH phosphatase on hMSCs differentiation into CMs.6、Karyotype analysis showing that Cr group(hMSCs which were not transfected), Scr (hMSCs which were transfected with vacant vector) and SSH group(hMSCs which were transfected with vector encoding SSH-1L) all maintained stable and normal diploid nucleus type without transform.7、During hMSCs differentiate into CMs induced by 5–Aza for four weeks , their morphology change was observed .Compared to the Cr group and Scr group,SSH group hMSCs showed multiple branches after one week, connecting with adjoining cells forming myotube-like structures after 2~3weeks and the myotube-like structures increased strikingly after 3~4weeks.On the basis of these experiments,we conclude that SSH group hMSCs are more powerful in differentiating into CMs than the other two control groups.8、We detected the microfilament changes during hMSCs differentiate into CMs induced by 5– Aza after four weeks. Cr, Scr and SSH group hMSCs’microfilament are all like long straight fiber bundle along the cell’s major axis with regular arrangement without induced.After induced by 5– Aza for one week, Cr group and Scr group hMSCs’microfilament basically along the cell’s major axis but with irregular arrangement,while SSH group hMSCs’microfilament spread to all directions. After induced for 2~3weeks, Cr group and Scr group hMSCs’microfilament spread to all directions, while SSH group hMSCs’microfilament join to one another and the actin gather together forming like intercalated disk structures. After induced for 3~4weeks, Cr group and Scr group hMSCs’microfilament join to one another forming like myotube-like structures, while SSH group hMSCs’actin gather together in microfilament junctions and forming like intercalated disk structures increase dramatically. On the basis of these experiments,we draw the conclusion that SSH-1Lplay an important role in hMSCs differentiation into CMs through regulating the rearrangement of cytoskeleton.9、We then measured the hMSCs’action potential by current clamp and find that the hMSCs which were not induced by 5-Aza not exhibit action potential,while,those hMSCs after induced exhibit weak action potential and the action potential of SSH group is stronger than the other two control groups. 10、RT-PCR analysis of mRNA expression for cardiac-specific genes in hMSCs shows that the genes can’t be detected in Cr, Scr and SSH group hMSCs without induced by 5-Aza. After induced by 5– Aza for one week, hMSCs express GATA4 and cTnT, which are early phase makers of cardimyogenic lineage.While SSH group hMSCs began to expressβ-MHC andα-sarcomeric actin which are late markers of cardiac lineage. After induced by 5– Aza for two weeks,Cr and Scr group hMSCs began to expressβ-MHC andα-sarcomeric actin.The mRNA expression level for cardiac-specific genes increased along with the elongation of induced time and the cardiac-specific gene expression level is much higher in SSH group hMSCs than in the other two control groups.11、Western blot analysis showed that the expression of cardiac-specific protein can’t be detected in Cr, Scr and SSH group hMSCs without induced by 5-Aza. After induced by 5– Aza for one week, SSH group hMSCs began to express Desmin, known to be early markers of myogenic differentiation. After induced for two weeks, Cr and Scr group began to express Desmin and then SSH group hMSCs begin to express of cardiac-specific protein cTnⅠandα-sarcomeric actin. While,Cr and Scr group expressed cTnⅠandα-sarcomeric actin until induced for three weeks. The cardiac-specific protein expression increased with the elongation of induced time and the SSH group hMSCs protein expression level is much higher than the other two control groups.In short, this study systemically elucidate that 5-Aza can induce the differentiation of hMSCs into CMs in vitro. We confirmed that SSH-1L can efficiently promote MSCs’differentiation into CMs through regulating the rearrangement of cytoskeleton for the first time. Our work offers helpful theory support and laboratory basis for further investigate on the mechanisms of stem cells differentiate into CMs.更多还原