小鼠胚胎干细胞的体外培养及向成骨细胞的转化

发布时间：2018-03-15 23:35

本文选题：胚胎干细胞　切入点：诱导　出处：《山西医科大学》2006年硕士论文　论文类型：学位论文

【摘要】： 目的:系统的研究小鼠胚胎干细胞的提取、体外培养扩增和向成骨细胞定向诱导分化。方法:用13.5d左右的昆明小鼠胚胎,采用常温消化法制备小鼠胚胎原代成纤维细胞(PMEF),加入专用的DMEM培养液,经丝裂霉素C处理后成为饲养层。在昆明小鼠受精后第4天,从子宫中取囊胚进行体外培养,4～5天后,获得大的巢式生长集落,使用0.25%胰蛋白酶-0.02% EDTA,采用两步离散法,对内细胞团集落进行离散,接种在新鲜制备的饲养层体系中,加入适宜的含有白血病抑制因子(LlF)的培养基进行分化抑制培养,并进行传代和体外扩增。采用碱性磷酸酶染色、核型分析、类胚体实验对扩增细胞进行ESC鉴定。将小鼠5d龄ES-D3细胞源的类胚体(EBs)单细胞,按5×104/mL接种12孔细胞培养板,在DMEM基础培养基中使EBs单细胞贴壁培养。于第14～21天时,分别添加50μg/mL维生素C、50 mmol/Lβ-磷酸甘油、1μmol/L地塞米松(A组), 50μg/mL维生素C、50 mmol/Lβ-磷酸甘油、1μmol/L地塞米松、10ng/mL骨形成蛋白2(B组),并设不添加诱导剂对照组(C组)。第22天时用1%茜素红染色显示阳性细胞,计算成骨细胞诱导形成率,数据用方差分析和多重比较检验。结果:成纤维细胞接种半小时后贴壁,可见细小突起长出,细胞呈梭形,核圆形。24小时后即可连生。ES细胞推开饲养层细胞生长,形成集落,集落边缘光滑清晰,有的集落边缘可见小的散在的ES细胞。细胞呈团状紧密排列,内部单个细胞辨别不清,每个细胞呈圆形,部分呈梭形,体积小,核大,胞浆少。细胞碱性磷酸酶染色呈强阳性,为二倍体核型,可形成类胚体。表明培养扩增后的细胞具有ESC的典型特征。在EBs单细胞贴壁培养的第14～21d加入成骨诱导剂后,细胞形态发生改变,原来的球形细胞逐渐发出突起,变为多角形或短梭形的类成纤维细胞样,茜素红染色为红色,证明已转化为成骨细胞。结论:1.原代成纤维细胞分离方法简单,来源方便,细胞贴壁快,增殖迅速,作为饲养层能促进ES细胞的增殖并抑制其分化,因而非常适用于ES细胞的分离培养。2.维生素C、β-磷酸甘油、地塞米松、BMP-2组成的成骨诱导剂能够诱导ESC分化为成骨细胞,诱导分化率为29%左右。
[Abstract]:Aim: to study systematically the extraction of mouse embryonic stem cells (ESCs), culture and amplification in vitro and differentiation into osteoblasts. Methods: Kunming mouse embryos of about 13.5 days were prepared by normal temperature digestion method. The primary fibroblasts of mouse embryos were prepared by normal temperature digestion method. The primary fibroblasts of mouse embryos were cultured in a special DMEM culture medium and then treated with mitomycin C to form a feeder layer after fertilization in Kunming mice on the 4th day after fertilization. After the blastocysts were cultured in vitro for 5 days, a large nest growth colony was obtained. 0.25% trypsin -0.02% EDTA was used to disperse the inner cell colony, and the colony was inoculated in the fresh feeder layer system. Suitable culture medium containing leukemia inhibitor LlF was added for differentiation inhibition culture, passage and in vitro amplification. Alkaline phosphatase staining and karyotype analysis were used. ESC identification of expanded cells was carried out by embryoid body experiment. Single cells of ES-D3 cells derived from ES-D3 cells of 5 days old in mice were inoculated with 12 well cell culture plate according to 5 脳 10 4 / mL, and EBs single cells were cultured in DMEM basic medium on day 14 to 21, respectively. 50 渭 g / mL vitamin C + 50 mmol/L 尾 -phosphoglycerol 1 渭 mol/L dexamethasone A group and 50 渭 g / mL vitamin C cor 50 mmol/L 尾 -phosphate glycerol phosphate 1 渭 mol/L dexamethasone 1 渭 mol/L dexamethasone 10 渭 mol/L / mL bone morphogenetic protein 2ng / mL group B were added respectively, and the control group C was stained with 1% alizarin red at 22 days after adding 50 渭 g / mL vitamin C and 10 渭 mol/L dexamethasone / mL bone morphogenetic protein 2ng / mL. Color positive cells, The osteoblast induction rate was calculated by ANOVA and multiple comparison test. Results: the fibroblasts adhered to the wall half an hour after inoculation, and the cells were spindle-shaped. After 24 hours of nuclear circle, they could push the feeder layer cells to grow, forming a colony, and the margin of the colony was smooth and clear. Small scattered es cells were seen on the edge of the colony. The cells were clustered and closely arranged, and the single cells were indistinguishable. Each cell was round, partly fusiform, small in size, large in nucleus, small in cytoplasm, and strongly positive for alkaline phosphatase staining. It is a diploid karyotype, which can form embryoid body. It shows that the expanded cells have the typical characteristics of ESC. After adding osteogenic inducer at 1421 days after EBs single cell adherent culture, the morphology of the cells changes and the original spherical cells gradually emit processes. It became polygonal or fusiform fibroblast-like, and alizarin red staining was red, which proved that it had been transformed into osteoblast. Conclusion the primary fibroblast isolation method is simple and convenient, the cells adhere to the wall quickly and proliferate rapidly. As the feeder layer, it can promote the proliferation and inhibit the differentiation of es cells. The osteogenic inducer composed of vitamin C, 尾 -glycerol phosphate and dexamethasone BMP-2 can induce the differentiation of ESC into osteoblasts and the differentiation rate is about 29%.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R329.2

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