胰腺导管上皮细胞分化为胰岛内分泌细胞的研究

发布时间：2018-01-13 11:38

本文关键词：胰腺导管上皮细胞分化为胰岛内分泌细胞的研究　出处：《暨南大学》2006年硕士论文　论文类型：学位论文

【摘要】：目的：分离、纯化、扩增大鼠胰腺导管上皮细胞，将其诱导分化为胰岛样细胞团，与天然胰岛比较葡萄糖刺激下的胰岛素和C肽分泌功能，为胰岛移植的临床应用提供可替代资源，并进一步完善干细胞培养技术，为今后的干细胞生物工程提供大量的细胞资源。方法：(1)分离大鼠胰腺，用差异性贴壁、低血清培养等方法纯化出大鼠胰腺导管上皮细胞，通过含KGF、EGF、ITS等细胞因子的基础培养基使其有效扩增，观察细胞增殖能力，并绘制生长曲线，免疫细胞化学染色法鉴定其表面标志ck-19的表达，RT-PCR检测PDX-1和insulin的表达。当细胞扩增、融合至80-90％时，改变细胞培养方案，通过含有exendin-4、KGF、NIC等细胞因子和葡萄糖的诱导分化培养基，对胰腺导管上皮细胞进行向胰岛细胞团转分化的实验。(2)观察细胞生长状态的变化并摄片。4周后对转分化后的细胞进行鉴定：DTZ染色；RT-PCR检测Glut-2、PDX-1、insulin、glucagon基因的表达；行葡萄糖刺激试验，用RIA法检测其胰岛素和C肽的分泌功能。(3)分离纯化大鼠天然胰岛，用同样的方法检测胰岛素和C肽的分泌功能，并与诱导转分化所得细胞团进行比较。结果：(1)成功分离、纯化了大鼠胰腺导管上皮细胞，并使其得到有效扩增，有效抑制了胰岛细胞和成纤维细胞的污染，，纯化后大部分细胞表达ck-19(胰腺导管上皮细胞的标志)，而RT-PCR显示PDX-1、insulin表达阴性。(2)诱导后细胞逐渐增大、变形，并出现类圆形小细胞，聚集成团，继而得到圆形或椭圆形的胰岛样团状结构，细胞团慢慢增大、增多，4周后细胞团DTz染色阳性，RT-PCR显示Glut-2、PDX-1、insulin、glucagon表达阳性。(3)对培养液上清的检测，诱导转分化所得胰岛细胞团在高糖刺激下的胰岛素分泌量为低糖时的6倍，在低浓度(3.3mM)和高浓度(16.7mM)时，诱导后胰岛细胞团胰岛素的分泌量分别约为天然胰岛的1／30和1／35。C肽的分泌量亦可随葡萄糖浓度的升高而增加，在低浓度和高浓度时分别为天然胰岛的1／32和1／26。结论：大鼠胰腺导管上皮细胞在体外可得到有效扩增并具有干细胞潜能，在细胞因子的作用下，可以进一步转分化为胰岛样细胞团，该胰岛样细胞团可随葡萄糖浓度的高低调节胰岛素和C肽的分泌。但与天然胰岛相比，胰岛素和C肽的分
[Abstract]:Objective: to isolate, purify, amplify and differentiate rat pancreatic ductal epithelial cells into islet like cell clusters. It can provide alternative resources for clinical application of islet transplantation, and further improve stem cell culture technology, and provide a large number of cell resources for future stem cell bioengineering. Methods Rat pancreatic ductal epithelial cells were isolated from rat pancreas by different adherent and low serum culture methods. The pancreatic ductal epithelial cells were purified by KGFGF-EGF. The basic medium of ITS and other cytokines can effectively amplify it, observe the ability of cell proliferation, draw the growth curve, and identify the expression of ck-19, the surface marker, by immunocytochemical staining. RT-PCR was used to detect the expression of PDX-1 and insulin. When the cells were amplified and fused to 80-90%, the cell culture was changed by exendin-4KGF. NIC and other cytokines and glucose induced differentiation medium. The pancreatic ductal epithelial cells were transdifferentiated into islet cell clusters. The changes of cell growth state were observed and the differentiated cells were identified by w-DTZ staining after 4 weeks. The expression of glucagon gene was detected by RT-PCR. The insulin and C-peptide secretion function was detected by RIA method. The natural islets of rats were isolated and purified. The secretory function of insulin and C-peptide was detected by the same method. And compared with the cell mass induced by transdifferentiation. Results 1) the pancreatic ductal epithelial cells of rats were successfully isolated and purified, and the pancreatic duct epithelial cells were effectively amplified, and the contamination of islet cells and fibroblasts was effectively inhibited. After purification, most of the cells expressed ck-19 (the marker of pancreatic ductal epithelial cells, but RT-PCR showed PDX-1 insulin expression negative.) after induction, the cells gradually increased. The round and oval islet like clusters were formed and the round or oval islet like structures were formed. The cell clusters increased slowly and the cell clusters were positive for DTz staining after 4 weeks. RT-PCR showed that Glut-2P PDX-1 insulin A glucagon positive expression. 3) the supernatant of culture medium was detected. The insulin secretion of islet cells stimulated by high glucose was 6 times as much as that of low glucose concentration (3.3 mm) and high concentration (16.7 mm). The insulin secretion of islet cell mass after induction was about 1/30 and 1 / 35. C peptide of natural islet, respectively, and increased with the increase of glucose concentration. At low and high concentrations, it was 1/32 and 1 / 26 of the natural islet, respectively. Conclusion: rat pancreatic ductal epithelial cells can be effectively expanded and have stem cell potential in vitro, and can be further transformed into islet like cell clusters under the action of cytokines. The islet like cell cluster can regulate the secretion of insulin and C-peptide with the glucose concentration, but compared with the natural islets, the insulin and C-peptide can be classified.
【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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