仿生电刺激在心肌细胞诱导胚胎干细胞向心肌样细胞分化中作用的研究

发布时间：2018-02-01 07:16

  本文关键词： 电刺激 胚胎干细胞 心肌细胞 共培养 细胞分化　出处：《重庆医科大学》2009年硕士论文　论文类型：学位论文

【摘要】： 目的探讨仿生电刺激对离体心肌微环境诱导胚胎干细胞(ESCs)向心肌样细胞分化的作用,为建立一套简单实用的胚胎干细胞移植技术提供理论依据。 方法分离培养SD大鼠乳鼠心肌细胞,以DAPI(4’6-联眯-2-苯基吲哚)染核作为细胞标记。收集昆明小鼠3.5d和4d胚龄的胚胎,以鼠间充质干细胞为饲养层,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,按是否给予胚胎干细胞仿生电刺激及心肌微环境诱导分为对照组、电刺激组、心肌组、电刺激+心肌细胞条件培养液组、电刺激+心肌组,每组又以实验干预培养时间(5、7、14天)分为3个亚组(Ⅰ、Ⅱ、Ⅲ组)。分别于实验的5、7、14天观察各组细胞生长情况,行肌钙蛋白T(cTnT)免疫荧光染色检测。 结果(1)以间充质干细胞作为饲养层细胞分离培养的昆明小鼠胚胎干细胞(ESCs)有其典型的形态学特征,对ESCs碱性磷酸酶进行检测,ES细胞显微镜下为棕褐色;Oct-4染色阳性。(2)电刺激组及对照组培养体系中始终未出现节律搏动的心肌样细胞,分化的细胞cTnT蛋白荧光染色均阴性;(3)心肌组、电刺激+心肌细胞条件培养液组、电刺激+心肌组均约7天左右出现节律搏动的心肌样细胞,观察至14天时,搏动频率约为30～50次/min。(4)电刺激+心肌细胞组和肌细胞、电刺激+心肌细胞条件培养液组相比,电刺激心肌细胞组中心肌细胞长势更好,ESCs分化而来的心肌样细胞搏动更快,更趋于一致性,记录已搏动和未搏动ESCs诱导分化后的心肌样细胞cTnT蛋白荧光染色阳性率更高,约为40%,显著高于心肌细胞组(28.7%)、电刺激+心肌细胞条件培养液组(17.1%)(P0.05)。 结论(1)以间充质干细胞作为饲养层细胞分离培养昆明小鼠胚胎干细胞可行,得到的胚胎干细胞能保持特殊的形态和未分化状态。(2)未经建系操作的ESCs可被诱导分化为节律搏动的心肌样细胞;成熟心肌细胞与ESCs共培养状态下,成熟心肌细胞可诱导ESCs向心肌细胞分化,这种微环境下ESCs向心肌细胞定向分化率显著高于自然分化组,即成熟心肌细胞是诱导ESCs定向分化中较强的诱导因素。(3)单独仿生电刺激因素还不足以诱导胚胎干细胞向心肌细胞定向分化。(4)仿生电刺激可促进心肌微环境诱导胚胎干细胞向心肌样细胞分化,提高胚胎干细胞定向分化为心肌样细胞的分化率,但是不能使胚胎干细胞向心肌样细胞分化的时间点提前。
[Abstract]:Objective to investigate the effect of bionic electrical stimulation on the differentiation of embryonic stem cells (ESCs) into cardiomyoid cells induced by isolated myocardial microenvironment, and to provide a theoretical basis for the establishment of a simple and practical technique for embryonic stem cell transplantation. Methods the neonatal rat cardiomyocytes were isolated and cultured, and the nuclei of DAPI4 (6-dime-2-phenylindole) were used as cell markers. The embryos of Kunming mice were collected at 3. 5 and 4 days of embryo age. Mouse mesenchymal stem cells (MSCs) were used as feeding layer for 5 days. The inner cell clusters were isolated and cultured. The colony growth was observed and stained by alkaline phosphatase (ALP). OCT-4 staining was used to identify the cell colonies, and then three or four generations of ESCs were selected and divided into control group, electrical stimulation group and myocardial group according to whether embryonic stem cells were given bionic electrical stimulation and myocardial microenvironment induction. Electrical stimulation of cardiomyocyte conditioned medium group and electrical stimulation group were divided into three subgroups (鈪，

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