体外诱导胰腺干细胞分化为胰岛样结构的初步探讨

发布时间：2018-03-13 11:26

本文选题：胰腺干细胞　切入点：诱导分化　出处：《广西医科大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的:优化胰腺干细胞原位传代扩增培养方法和技术,建立并形成稳定的胰腺干细胞系,获得较大量胰腺干细胞并体外诱导分化为胰岛样结构。方法:优化原位传代扩增培养的方法对胰腺干细胞进行扩增,建立胰腺干细胞系。选择生长状态良好的的第二代胰腺干细胞分为四组:空白对照组、葡萄糖组、尼克酰胺组、血清尼克酰胺组,体外连续诱导培养4周,观察胰腺干细胞的形态学特征变化规律;以DTZ染色、Mallory染色对诱导后获得的胰岛样结构行进行初步鉴定;胰岛素抗体免疫荧光组织化学染色方法,检测所诱导的胰岛样结构中的胰岛素阳性细胞。结果:空白对照组:胰腺干细胞随着培养时间的延长,细胞形态及生长方式发生改变,由原来的圆形、立体感强变为扁平、折光性降低、立体感减弱;葡萄糖诱导组:诱导9天后,胰腺干细胞聚集,成串样生长,诱导20天后,细胞变扁平,未形成胰岛样结构;尼克酰胺诱导组:诱导5天后,胰腺干细胞逐渐靠拢,诱导25天后,胰腺干细胞形成椭圆形结构;血清尼克酰胺组:诱导1周后,胰腺干细胞逐渐靠拢,汇集成团,诱导2周后,汇集的细胞团增大,但未形成圆形或者椭圆形的结构,诱导3周后,细胞团逐渐形成有完整包膜包裹的胰岛样结构;胰岛样结构DTZ染色,呈棕红色;Mallory染色可见胰岛样结构中周围的细胞呈红色,中央的细胞呈淡黄色;收集胰岛样结构,制作石蜡切片后行Mallory染色,可见有橘黄色、红色两种细胞;胰岛素抗体免疫荧光组织化学染色,在紫外光线激发下,检测到呈绿色荧光的胰岛素阳性细胞。结论:(1)优化后的原位传代扩增培养方法,可扩增并建立胰腺干细胞系,获得较多数量的胰腺干细胞,满足后续实验的需要。(2)在血清尼克酰胺诱导条件下,胰腺干细胞可分化为有完整包膜包裹、DTZ染色阳性、Mallory染色可见两种细胞、胰岛素抗体免疫荧光组织化学染色检测到阳性细胞的胰岛样结构。
[Abstract]:Aim: to optimize the method and technique of in situ culture of pancreatic stem cells, to establish and form stable pancreatic stem cell lines, to obtain a large number of pancreatic stem cells and to induce and differentiate into islet like structures in vitro. Methods: pancreatic stem cell lines were established by optimizing the method of in situ passage and culture. The second generation pancreatic stem cells, which grew well, were divided into four groups: blank control group, glucose group and nicotinamide group. Serum nicotinamide group was cultured for 4 weeks in vitro to observe the morphological characteristics of pancreatic stem cells, and the islet like structure was preliminarily identified by DTZ staining. Insulin positive cells in islet-like structure were detected by immunofluorescence histochemical staining. Results: in the blank control group, with the extension of culture time, the morphology and growth pattern of pancreatic stem cells changed from round, strong stereosensory to flat, reduced refraction and reduced stereosensory. Glucose-induced group: after 9 days of induction, pancreatic stem cells gathered and grew in strands. After 20 days of induction, the cells flattened and did not form islet structure, while in nicotinamide group, pancreatic stem cells gradually converged after 5 days of induction. After 25 days of induction, pancreatic stem cells formed oval structure, serum-nicotinamide group: after 1 week of induction, pancreatic stem cells gradually converged to form clusters, and after 2 weeks of induction, the aggregation of pancreatic stem cells increased. However, the round or oval structure was not formed. After 3 weeks of induction, the islet like structure was gradually formed, and the DTZ staining of islet like structure showed that the cells around the islet like structure were red. The cells in the center were yellowish; the islet-like structures were collected, and the paraffin sections were made with Mallory staining. There were two kinds of cells, orange and red; insulin antibodies were stained by immunofluorescence histochemistry, stimulated by ultraviolet rays. Insulin positive cells with green fluorescence were detected. Conclusion the optimized in situ culture method can amplify and establish pancreatic stem cell lines and obtain a large number of pancreatic stem cells to meet the needs of subsequent experiments. Under the condition of serum nicotinamide induction, a large number of pancreatic stem cells can be obtained. Pancreatic stem cells could be differentiated into two kinds of cells with intact encapsulated DTZ positive staining. The islet like structure of the positive cells was detected by insulin antibody immunofluorescence histochemical staining.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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