体外定向诱导小鼠胚胎干细胞向血管平滑肌细胞分化

发布时间：2018-02-23 19:47

  本文关键词： 胚胎干细胞 血管平滑肌细胞 血小板源性生长因子　出处：《南昌大学》2008年硕士论文　论文类型：学位论文

【摘要】： 目的:通过选择适当的诱导剂,探讨胚胎干细胞体外向血管平滑肌细胞分化的趋势。观察胚胎干细胞在体外不同条件下定向诱导分化血管平滑肌细胞的能力。 方法:实验于2006年8月至2007年2月在南昌大学第二附属医院血液病研究所完成。①动物及细胞系:清洁级孕12.5天的昆明小白鼠1只,实验过程中对动物的处置符合动物伦理学标准;小鼠胚胎干细胞系129X/SvJ由美国ATCC提供。②实验方法:从12.5天的胎鼠中分离出原代胚胎成纤维细胞,当原代胚胎成纤维细胞传至3～5代时,更换为含体积分数为0.15胎牛血清的DMEM高糖培养基,24小时后收集培养液,加入条件培养基。用其对小鼠胚胎干细胞进行体外培养,传代时用差速贴壁法分离去除已分化的胚胎干细胞,经过悬滴-悬浮培养,构建拟胚体分化模型。设立3组,各组均置于0.1%明胶处理过的T25培养瓶中,每瓶加入50个拟胚体,均匀分布于培养瓶底,使拟胚体贴壁生长。诱导组7～10天加入10-9mol/L全反式维甲酸和3μg/L转化生长因子β1,10～21天加入20μg/L血小板源性生长因子,血清对照组仅加入去生长因子胎牛血清,全反式维甲酸组加入全反式维甲酸。诱导21天的拟胚体,用胰蛋白酶和胶原酶Ⅱ联合消化为单个细胞,再加入20μg/L血小板源性生长因子继续诱导7天。③实验评估:应用逆转录-聚合酶链反应(RT-PCR)检测诱导细胞血管平滑肌肌动蛋白、血管平滑肌肌球蛋白重链基因的表达,通过免疫细胞化学技术检测血管平滑肌肌动蛋白的表达来鉴定细胞性质。 结果:①胚胎干细胞生长及拟胚体诱导分化:胚胎干细胞在体外能自发形成拟胚体,经过不同生长因子的分阶段联合诱导,拟胚体周围出现大量的梭形细胞。②RT-PCR检测:拟胚体血管平滑肌肌动蛋白和血管平滑肌肌球蛋白重链基因均呈阳性表达。③免疫细胞化学检测:荧光显微镜下,罗丹明染色细胞浆呈红色荧光,并可见肌丝结构;DAPI染色细胞核呈蓝色荧光。诱导组血管平滑肌肌动蛋白阳性率明显高于血清对照组和全反式维甲酸组(P0.01)。 结论:①在条件培养基无饲养层上可大量扩增胚胎干细胞并保持其未分化状态。②胚胎干细胞经全反式维甲酸、转化生长因子β1以及血小板源性生长因子分阶段联合诱导后,可分化为血管平滑肌细胞且纯度较高。
[Abstract]:Aim: to investigate the tendency of embryonic stem cells to differentiate into vascular smooth muscle cells (VSMCs) in vitro by selecting suitable inducers, and to observe the ability of embryonic stem cells to induce differentiation of vascular smooth muscle cells (VSMCs) under different conditions in vitro. Methods: from August 2006 to February 2007, 1 animal and cell line were completed in the Institute of Hematology, second affiliated Hospital of Nanchang University. The treatment of animals in the course of the experiment was in accordance with the standard of animal ethics; the mouse embryonic stem cell line 129X / SvJ was provided by the ATCC of the United States with the method of 2.2. Primary embryonic fibroblasts were isolated from 12.5-day-old fetal mice. When the primary embryonic fibroblasts were transferred to 3 ~ 5 passages, the DMEM medium containing 0.15 fetal bovine serum was replaced with high sugar medium for 24 hours, then the medium was collected and added to the conditioned medium. The mouse embryonic stem cells were cultured in vitro. The differentiated embryonic stem cells were separated and removed by differential adherent method during passage. The model of embryoid differentiation was established by suspension and suspension culture. Three groups were set up and each group was placed in the culture flask of T25 treated with 0.1% gelatin, 50 embryoid bodies were added to each bottle. In the induction group, 10-9 mol / L all-trans retinoic acid and 3 渭 g / L transforming growth factor 尾 _ 1 / L were added to the platelet-derived growth factor for 10 ~ 21 days, while the serum control group was added only to the bovine serum derived from growth factor. The all-trans retinoic acid group was added with all-trans retinoic acid. The embryoid body was induced for 21 days and digested into a single cell by trypsin and collagenase 鈪，

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