体外模拟心肌微环境下胚胎干细胞向心肌样细胞分化的实验研究

发布时间：2018-05-02 03:19

  本文选题：胚胎干细胞 + 共培养　； 参考：《重庆医科大学》2008年硕士论文

【摘要】： 目的:探讨完整成熟心肌细胞或心肌细胞裂解物对胚胎干细胞(embryonic stem cells,ESCs)定向分化为心肌细胞的诱导作用及强度。 方法:收集昆明小鼠3.5d和4d胚龄的囊胚,分别将其培养在小鼠胚胎成纤维细胞饲养层上,4～5d后取隆起生长的内细胞团分离后再培养,观察集落的生长情况并通过碱性磷酸酶染色、OCT-4染色等对细胞集落进行鉴定。然后取3～4代ESCs,先将ESCs悬浮培养2～3d形成拟胚体(embryoid bodies,EBs),再用大鼠心肌细胞等诱导因素对其诱导培养。共分5组进行定向诱导实验,即心肌细胞诱导组、心肌细胞条件培养液诱导组、心肌细胞裂解液诱导组、胚胎干细胞分化培养液诱导组和对照组。ESCs分化为心肌细胞的结果判断,用相差显微镜观察EBs搏动情况,用免疫细胞荧光技术检测心肌细胞特异性肌钙蛋白T(TnT)、а-肌动蛋白(а-Actin)的表达。 结果:①采用0.1%胰蛋白酶-0.02%EDTA室温作用2-4分钟并辅以机械作用,分离克隆ESCs效果较好。②心肌细胞诱导组:诱导分化培养第3天起有EBs自发性、有节律跳动,12d时有93%的EBs出现节律性收缩。已分化的ESCs特异性肌钙蛋白T、а-Actin阳性表达率56.5±13.44%,显著高于其它各组(P0.05)。③心肌细胞条件培养液诱导组:12d时有8%的EBs出现节律性收缩。已分化的ESCs肌钙蛋白T、а-Actin阳性表达率5.2±2.01%,显著低于心肌细胞诱导组(P0.05),与对照组比较差异无显著性(P0.05)。④心肌细胞裂解液诱导组:12d时有36%的EBs出现节律性收缩。已分化的ESCs特异性肌钙蛋白T、а-Actin阳性表达率8.2±2.45%,显著低于心肌细胞诱导组(P0.05),显著高于对照组(P0.05)。⑤胚胎干细胞分化培养液诱导组:12d时有7%的EBs出现节律性收缩。已分化的ESCs特异性肌钙蛋白T、а-Actin阳性表达率5.08±1.38%,显著低于心肌细胞诱导组(P0.05),与对照组比较差异无显著性(P0.05)。 结论:①采用4d胚龄的囊胚和0.1%胰蛋白酶-0.02%EDTA室温作用2-4分钟并辅以机械作用的消化方法有助于提高昆明小鼠ES细胞建株能力。②心肌细胞和心肌细胞裂解液均可诱导ESCs向心肌细胞定向分化,且心肌细胞的诱导作用强于心肌细胞裂解液。
[Abstract]:Aim: to investigate the induction effect and intensity of intact mature cardiomyocytes or cardiomyocyte lysates on differentiation of embryonic stem cells (embryonic stem cells) into cardiomyocytes. Methods: the blastocysts of Kunming mice were collected for 3.5 days and 4 days old respectively. The blastocysts were cultured on the feeder layer of mouse embryonic fibroblasts for 4 days. The colony growth was observed and the colony was identified by alkaline phosphatase staining and OCT-4 staining. ESCs suspension culture was carried out for 2 days to form embryoid bodies-ebs, and then was induced by rat cardiomyocytes. Five groups were divided into five groups: cardiomyocyte induction group, cardiomyocyte conditioned medium group, cardiomyocyte lysis medium induction group, embryonic stem cell differentiation culture medium induction group and control group. ESCs differentiated into cardiomyocytes. The EBs pulsation was observed by phase contrast microscope, and the expression of TnTnTnTnTnTnTnTnTX, actin was detected by immunocytofluorescence technique. Results using 0.1% trypsin and 0.02TA at room temperature for 2 to 4 minutes, the cloned ESCs was isolated and cloned. 2 the cardiomyocyte induction group. 2. In the cardiomyocyte induction group, there was spontaneous EBs from the third day after induced differentiation and culture. At 12 days, 93% of EBs showed rhythmic contraction. The positive rate of differentiated ESCs specific troponin T and tactin was 56.5 卤13.44, which was significantly higher than that of the other groups (P 0.05n.3). 8% of EBs showed rhythmic contraction at 12 days in the conditioned medium group. The positive expression rate of ESCs troponin Tand activity was 5.2 卤2.01, which was significantly lower than that in cardiomyocyte induction group (P 0.05), but there was no significant difference between the two groups. 36% of EBs showed rhythmic contraction at 12 days after being induced by lytic solution of cardiomyocytes. The positive expression rate of ESCs specific troponin Tand activity was 8.2 卤2.45, which was significantly lower than that in cardiomyocyte induction group (P 0.05), and was significantly higher than that in control group (P 0.05.5). 7% of EBs showed rhythmic contraction at 12 days in the control group induced by P0.055 embryonic stem cell differentiation medium. The positive expression rate of ESCs specific troponin Tand activity was 5.08 卤1.38, which was significantly lower than that in cardiomyocyte induced group (P 0.05), and there was no significant difference compared with the control group (P 0.05). Conclusion the digestion of the blastocyst with 4 d embryo age and 0.1% trypsin-0.02TA at room temperature for 2 to 4 minutes, supplemented by mechanical action, is helpful to improve the ability of mouse es cells to build 2 cardiomyocytes and cardiomyocyte lytic fluid. All of them could induce ESCs to differentiate into cardiomyocytes. The inductive effect of myocardial cells was stronger than that of myocardial cell lysate.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

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