诱导小鼠多能干细胞分化为角膜上皮细胞的实验研究

发布时间：2018-01-01 21:26

本文关键词：诱导小鼠多能干细胞分化为角膜上皮细胞的实验研究　出处：《大连医科大学》2013年硕士论文　论文类型：学位论文

【摘要】：目的：探讨鼠源诱导多能干细胞(mice induced pluripotent stem cells，miPSCs）通过与小鼠角膜基质细胞共培养途径诱导分化为角膜上皮的可行性，检测miPSCs向角膜上皮细胞分化后形态学变化以及部分标志蛋白表达的变化，为研究miPSCs向角膜上皮的分化提供实验依据。方法：购买小鼠ips细胞株和辐射后小鼠胚胎成纤维细胞株（mouse embryonicfibroblast MEF），使用饲养层细胞法培养扩增小鼠来源ips细胞。应用Western blotting法检测诱导分化前鼠源ips细胞多能性标志性蛋白Oct4、Nanog和Sox2的表达以进行鉴定。体外分离，增殖小鼠角膜基质细胞。将鼠源iPSCs在体外环境与小鼠角膜基质细胞共培养以诱导分化。应用倒置显微镜观察诱导分化1周后miPSCs的形态学变化，并应用免疫荧光法检测miPSCs分化后角膜上皮标志蛋白K12的表达变化，RT-PCR法检测细胞分化后K12的mRNA表达。结果：培养的miPSCs复苏3天后在倒置显微镜下观察呈克隆样生长，，克隆呈圆形或椭圆形，边界清楚。克隆内细胞排列紧密，细胞体积小，核大，核仁清晰，细胞核/质比高。应用Western blotting对其miPSCs进行鉴定，结果显示细胞均表达多能性标志性蛋白Oct4、Nanog和Sox2；分化后细胞呈多角形，胞体发暗，排列疏松，细胞核/质比变小。免疫荧光法检测分化前miPSCs的K12均未见表达，miPSCs经1周的诱导分化后其细胞均表达角膜上皮标志蛋白K12。结论：使用饲养层细胞法扩增小鼠来源ips细胞能够使其在扩增数代后仍然保留有多向分化潜能而不分化。在体外与小鼠角膜基质细胞共培养，能够将鼠源iPSCs细胞定向诱导分化成为角膜上皮样细胞，使miPSCs不仅从形态学上向角膜上皮分化，同时成功表达角膜上皮细胞标志蛋白K12。
[Abstract]:Objective: to study induced pluripotent stem cells in mice induced by murine pluripotent stem cells. The feasibility of inducing differentiation into corneal epithelium by co-culture with mouse corneal stromal cells (miPSCs). The morphological changes and the expression of some marker proteins after the differentiation of miPSCs into corneal epithelial cells were detected to provide experimental basis for studying the differentiation of miPSCs into corneal epithelium. Methods: the mouse ips cell line and the irradiated mouse embryonicfibroblast MEF cell line were purchased. Murine ips cells were cultured and amplified by feeder layer cell culture and Western blotting method was used to detect the multipotent marker protein Oct4 of mouse ips cells before induction of differentiation. Expression of Nanog and Sox2 for identification. Isolated in vitro. Mouse corneal stromal cells were proliferated. Mouse iPSCs was co-cultured with mouse corneal stromal cells in vitro to induce differentiation. The morphological changes of miPSCs after 1 week of differentiation were observed by inverted microscope. . The expression of corneal epithelial marker protein K12 after miPSCs differentiation was detected by immunofluorescence assay. The mRNA expression of K12 was detected by RT-PCR. Results: after 3 days of resuscitation, the cultured miPSCs were observed to grow like clones under inverted microscope, the clones were round or elliptical, the boundaries were clear, the cells in the clones were arranged tightly, the cells were small and the nuclei were large. The nucleoli were clear and the ratio of nucleus to cytoplasm was high. The miPSCs was identified by Western blotting. The results showed that all cells expressed multipotent iconic protein Oct4. Nanog and Sox2; After differentiation, the cells showed polygonal shape, dark cell body, loose arrangement and smaller nuclear / cytoplasm ratio. No expression of K12 in miPSCs was detected by immunofluorescence. MiPSCs cells expressed corneal epithelial marker protein K12 after 1 week of differentiation. Conclusion: using feeder layer cell method to amplify murine ips cells can keep the ability of differentiation without differentiation and co-culture with mouse corneal stromal cells in vitro. Mouse iPSCs cells could be induced to differentiate into corneal epithelioid cells, and miPSCs not only differentiated into corneal epithelium morphologically, but also successfully expressed the corneal epithelial marker protein K12.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R772.2

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