混合饲养层培养的人胚胎干细胞向胰岛素分泌细胞的诱导分化

发布时间：2018-06-16 09:10

本文选题：人胚胎干细胞 + 胰岛素分泌细胞　；参考：《郑州大学》2011年硕士论文

【摘要】：糖尿病(diabetes mellitus, DM)是一组以慢性血葡萄糖水平增高为特征的代谢疾病群。糖尿病的病因尚未完全阐明,主要认为与遗传因素和环境因素等有关,口服降糖药和外源性胰岛素替代治疗是临床治疗的主要方法,但却无法防止晚期糖尿病并发症的产生。近年来胰腺或胰岛细胞移植作为目前治疗Ⅰ型糖尿病和部分Ⅱ型糖尿病效果最理想的方法不断受到关注。人胚胎干细胞(human embryonic stem cells, hESCs)是一种具有分化发育成三个胚层组织细胞潜能的全能性的细胞。利用hESCs的多向分化能力,将其定向诱导为胰岛素分泌细胞(insulin producing cells, IPCs)为糖尿病的细胞移植治疗提供了新的方向。目前虽已有文献报道hESCs成功诱导分化为胰岛素分泌细胞,但诱导分化效率偏低,成为胰岛素分泌细胞相关研究的主要障碍,故本研究通过经由拟胚体方法和贴壁培养方法及添加不同浓度诱导因子诱导混合饲养层培养体系的人胚胎干细胞定向分化为胰岛素分泌细胞进行对比,通过优化体外诱导条件,以期达到最佳的诱导分化效率。目的比较不同诱导分化方法对hESCs向胰岛素分泌细胞分化效率的影响。方法 1.实验材料为郑州大学第一附属医院生殖中心自主建立的hESCs系：ZZU(郑州大学)-hESC(人胚胎干细胞)-1,并在混合饲养层上培养。 2. hESCs诱导为限定性内胚层过程中,根据加入活化素A的浓度(为25ng/ml、50ng/ml和100ng/ml)不同分为3组。 3.hESCs诱导为胰岛素分泌细胞过程中,根据经拟胚体法和贴壁培养直接诱导法分为2组,诱导分化主要分为4个阶段。 4.采用Sox-17免疫荧光法测定限定性内胚层的表达,RT-PCR法检测各个阶段特异表达基因,诱导分化末行双硫腙染色和胰岛素释放实验鉴定分化成熟的胰岛样细胞团。 5.通过比较不同诱导方案的诱导效率,得出较好的诱导方案。结果 1.随着诱导分化时间的延长,可见细胞形态的变化。 2.Sox-17免疫荧光结果示,活化素A的浓度为50ng/ml组和100ng/ml组诱导分化效率无明显差异(P0.05),25ng/ml组诱导分化效率低于上述两组.(P0.05)。 3.根据诱导分化末双硫腙染色、RT-PCR鉴定和胰岛素释放试验的结果估算出：采用贴壁培养细胞直接进行诱导分化,可得到较多的胰岛素分泌细胞。结论 1、50ng/ml的活化素A同样可获得较多的限定性内胚层细胞,为后续的研究提供细胞。 2、采用贴壁培养hESCs直接进行4阶段诱导分化得到胰岛素分泌细胞的效率较高。
[Abstract]:Diabetes mellitus (DMM) is a group of metabolic diseases characterized by chronic elevated blood glucose levels. The etiology of diabetes has not been completely clarified, which is mainly related to genetic factors and environmental factors. Oral hypoglycemic drugs and exogenous insulin replacement therapy are the main methods of clinical treatment, but can not prevent the occurrence of complications in advanced diabetes mellitus. In recent years, pancreas or islet cell transplantation has attracted more and more attention as the most effective method for the treatment of type 1 diabetes and partial type 2 diabetes. Human embryonic stem cells (embryonic stem cells, hESCs) are totipotent cells with the potential to differentiate into three embryonic tissue cells. The induction of hESCs into insulin producing cells (IPCs) provides a new direction for the treatment of diabetes mellitus. Although it has been reported that hESCs have been successfully induced to differentiate into insulin-secreting cells, the efficiency of inducing differentiation is relatively low, which has become the main obstacle to the study of insulin secretory cells. In this study, human embryonic stem cells were induced to differentiate into insulin-secreting cells by embryoid method, adherent culture method and different concentration of induction factors, and the induction conditions in vitro were optimized. In order to achieve the best induction and differentiation efficiency. Objective to compare the effects of different differentiation induction methods on the differentiation efficiency of hESCs into insulin-secreting cells. Method 1. The experimental materials were the human embryonic stem cells (hESC) -1 and cultured on the mixed feeder layer. 2. The hESCs were induced to be limited endoderm in the process of inducing hESCs into the limited endoderm, which was established by the procreation Center of the first affiliated Hospital of Zhengzhou University. According to the concentration of activin A (25ng / ml 50ng / ml and 100ng / ml), the cells were divided into three groups. 3. During the induction of HESCs into insulin-secreting cells, they were divided into two groups according to the embryoid method and the direct induction method of adherent culture. Induction and differentiation are mainly divided into four stages. 4. Sox-17 immunofluorescence assay was used to detect the expression of restricted endoderm and RT-PCR was used to detect the specific expression genes in each stage. Dithizone staining and insulin release assay were used to identify the differentiated islet like cell clusters at the end of differentiation. 5. By comparing the induction efficiency of different induction schemes, a better induction scheme was obtained. Result 1. With the prolongation of induction and differentiation time, the changes of cell morphology were observed. 2. Sox-17 immunofluorescence results showed that, The concentration of activin A in 50ng/ml group and 100ng/ml group had no significant difference in inducing differentiation efficiency. The differentiation efficiency of 25 ng / ml group was lower than that of the above two groups. According to the results of RT-PCR identification and insulin release test, it was estimated that more insulin-secreting cells could be obtained by direct differentiation induced by adherent cells. Conclusion 1 50 ng / ml activin A can also obtain more limited endoderm cells. For the further study, the hESCs were directly induced to differentiate into insulin-secreting cells in four stages by adherent culture.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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