猪羊水干细胞特异向心肌细胞分化及其中Wnt通路相关因子的表达变化

发布时间：2019-06-20 22:51

【摘要】：羊水干细胞具有向三胚层细胞分化的能力,心肌细胞起源于中胚层,因此羊水干细胞也具有向心肌细胞分化的潜能,但目前的研究显示羊水干细胞向节律性心肌细胞分化的效率还相对较低。猪在生理和生化等性状上与人类具有很多的相似处,因此研究猪羊水干细胞向心肌细胞分化具有重要的临床应用价值。 1、心肌细胞诱导分化研究本研究以猪羊水干细胞为研究对象,用5-氮胞苷(5-aza)和维生素C(Vc)为诱导剂,对猪羊水干细胞形成的类胚体(EBs)进行诱导分化。应用免疫荧光、RT-PCR、透射电镜技术检测跳动细胞团中心肌特异性标记的表达情况。结果显示,在猪羊水干细胞形成的类胚体中加入心肌细胞诱导剂10 d后,有节律性跳动的细胞团出现,采用0.1 mmol/L Vc加5μmol/L 5-aza联合诱导组的诱导效率最高,出现约33%的跳动细胞团。免疫荧光结果显示,跳动心肌细胞团表达细胞骨架蛋白α-actin和肌钙蛋白Tnni3。RT-PCR检测跳动心肌细胞团发现,心肌细胞特异性标记分子TbX5、Gata4、α-MHC、Tnni3均呈阳性表达。借助透射电镜观察诱导后的跳动样细胞团,能清晰观察到肌丝、糖原粒、糖原池等结构。说明5-氮胞苷和维生素C可以促进猪羊水干细胞向心肌细胞的诱导分化,从而建立了一种有效诱导猪羊水干细胞向心肌细胞分化的方法。 2、Wnt/β-catenin信号通路在心肌细胞诱导分化中的作用为了进一步探索猪羊水干细胞向心肌细胞分化的机理,本研究用经典Wnt信号通路的激活剂和抑制剂处理向心肌细胞分化的pAFS细胞,运用RT-PCR、实时定量PCR、免疫荧光等检测手段,研究了经典Wnt信号通路在pAFS细胞向心肌细胞分化过程中的调控作用,结果显示Wnt/β-catenin信号通路在一定程度上能够促进pAFS向心肌细胞分化,对心肌细胞诱导分化的机制进行了初步探索。
[Abstract]:Amniotic fluid stem cells have the ability to differentiate into triembryonic cells, and cardiomyocytes originate from mesoderm, so amniotic fluid stem cells also have the potential to differentiate into cardiomyocytes. However, current studies have shown that amniotic fluid stem cells can differentiate into rhythmic cardiomyocytes. Pigs have many similarities with human in physiological and biochemical traits, so it is of great clinical value to study the differentiation of pig amniotic fluid stem cells into cardiomyocytes. 1. In this study, pig amniotic fluid stem cells were used as the object of study. 5-azacytidine (5-aza) and vitamin C (Vc) were used as inducers to induce the differentiation of embryoid (EBs) formed by pig amniotic fluid stem cells. The expression of muscle specific markers in the center of beating cell mass was detected by immunofluorescence and RT-PCR, transmission electron microscope. The results showed that 10 days after adding cardiomyocyte inducer to the embryoid formed by pig amniotic fluid stem cells, the rhythmic beating cell mass appeared. The induction efficiency of 0.1 mmol/L Vc plus 5 渭 mol / L 5-aza was the highest, and about 33% of the beating cell mass appeared. The results of immunofluorescence showed that the expression of cytoskeleton protein 伪-actin and troponin Tnni3.RT-PCR were positive in beating cardiomyocytes. TbX5,Gata4, 伪-MHC,Tnni3, a specific marker molecule, was found to be positive in beating cardiomyocytes. With the help of transmission electron microscope, the structure of myofilament, glycogen, glycogen pool and so on could be clearly observed by observing the induced beating-like cell mass. It is suggested that 5-azacytidine and vitamin C can promote the differentiation of pig amniotic fluid stem cells into cardiomyocytes, thus establishing an effective method to induce the differentiation of pig amniotic fluid stem cells into cardiomyocytes. 2. The role of WNT / 尾-catenin signaling pathway in the differentiation of cardiomyocytes in order to further explore the mechanism of differentiation of pig amniotic fluid stem cells into cardiomyocytes, pAFS cells differentiated into cardiomyocytes were treated with classical Wnt signal pathway activators and inhibitors, and RT-PCR, real-time quantitative PCR, immunofluorescence was used. The regulation of classical Wnt signaling pathway in the differentiation of pAFS cells into cardiomyocytes was studied. the results showed that Wnt/ 尾-catenin signaling pathway could promote the differentiation of pAFS into cardiomyocytes to a certain extent, and the mechanism of induction and differentiation of cardiomyocytes was explored.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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