胰腺干细胞分化的胰岛细胞与天然胰岛细胞基因差异研究

发布时间：2018-02-05 22:10

本文关键词： 胰腺导管干细胞转分化胰岛内分泌细胞天然胰岛基因表达谱　出处：《暨南大学》2006年硕士论文　论文类型：学位论文

【摘要】：目的：体外分离培养大鼠胰腺导管干细胞，并对其纯化、扩增、鉴定，转分化为胰岛内分泌细胞，分离纯化大鼠天然胰岛，通过基因表达谱芯片研究转分化的胰岛内分泌细胞与天然胰岛细胞在成熟度上的差异，探讨哪些基因对转分化起着关键性调节作用。方法：酶消化法分离大鼠胰腺导管干细胞，CK-19、PDX-1进行免疫细胞化学染色鉴定。密度梯度离心法分离纯化大鼠天然胰岛，，双硫腙染色鉴定转分化的胰岛内分泌细胞与天然胰岛细胞。对转分化的胰岛内分泌细胞进行葡萄糖刺激实验，ELISA法测定胰岛细胞的胰岛素分泌量，对诱导分化不同时间点的干细胞培养基进行C肽测定。采用大鼠22000位点寡核苷酸芯片对转分化的胰岛内分泌细胞和分离纯化的天然胰岛提取RNA样品进行3次杂交重复实验，筛选差异性表达的基因。结果：分离培养的大鼠胰腺导管干细胞经免疫组化染色呈CK-19、PDX-1染色阳性。经过诱导分化后，细胞形成类胰岛样细胞团，呈巢团样聚集，双硫腙染色为棕红色。体外分离的大鼠天然胰岛与转分化的胰岛内分泌细胞的基因表达谱芯片差异性分析发现一些参与胰岛发育的关键基因的表达明显下调。结论：体外培养条件下大鼠胰腺干细胞可增殖并转分化为胰岛样细胞，转分化的培养基中胰岛素分泌水平远低于天然胰岛细胞，表明转分化而来的胰岛内分泌细胞功能与天然胰岛仍存在较大差异。基因表达谱芯片研究发现转分化的胰岛内分泌细胞与天然胰岛细胞基因表达差异显著，证明转分化的胰岛内分泌细胞不成熟。
[Abstract]:Objective: to isolate and culture rat pancreatic ductal stem cells in vitro, purify, amplify, identify, differentiate into endocrine cells of pancreatic islets, and isolate and purify natural pancreatic islets of rats. Gene expression microarray was used to study the difference in maturity between transdifferentiation endocrine cells and natural islet cells, and to explore which genes play a key role in the transdifferentiation. Methods: CK-19 PDX-1 was isolated from rat pancreatic ductal stem cells by enzyme digestion and identified by immunocytochemical staining. The natural islets were isolated and purified by density gradient centrifugation. Dithizone staining was used to identify the transdifferentiation of islet endocrine cells and natural islet cells. The insulin secretion of the transformed islet endocrine cells was determined by Elisa. C-peptide assay was performed on stem cell culture medium at different time points to induce differentiation. RNA samples were extracted by rat oligonucleotide microarray at site 22000 on transdifferentiated islet endocrine cells and isolated and purified natural islets. Three repeated hybridization experiments were carried out. Screening for differentially expressed genes. Results: the isolated and cultured rat pancreatic ductal stem cells showed CK-19pPDX-1 positive staining by immunohistochemical staining. After induced differentiation, the cells formed islet like cell clusters. Gathered in nests. Dithizone was stained brown red. The gene expression microarray analysis showed that some key genes involved in islet development were down-regulated. Conclusion: rat pancreatic stem cells can proliferate and differentiate into islet like cells in vitro. The insulin secretion level in the transdifferentiation medium is much lower than that in natural islet cells. The results showed that the function of the transformed islet endocrine cells was still different from that of the natural islets, and the gene expression of the transformed islet endocrine cells was significantly different from that of the natural islet cells. The results showed that the differentiated islet endocrine cells were immature.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R329

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