本文關鍵詞：胰腺導管上皮細胞分化為胰島內分泌細胞的研究　出處：《暨南大學》2006年碩士論文　論文類型：學位論文

  更多相關文章： 胰腺干細胞 胰腺導管上皮細胞 誘導分化 胰島樣細胞團 糖尿病

【摘要】：目的：分離、純化、擴增大鼠胰腺導管上皮細胞，將其誘導分化為胰島樣細胞團，與天然胰島比較葡萄糖刺激下的胰島素和C肽分泌功能，為胰島移植的臨床應用提供可替代資源，并進一步完善干細胞培養(yǎng)技術，為今后的干細胞生物工程提供大量的細胞資源。 方法：(1)分離大鼠胰腺，用差異性貼壁、低血清培養(yǎng)等方法純化出大鼠胰腺導管上皮細胞，通過含KGF、EGF、ITS等細胞因子的基礎培養(yǎng)基使其有效擴增，觀察細胞增殖能力，并繪制生長曲線，免疫細胞化學染色法鑒定其表面標志ck-19的表達，RT-PCR檢測PDX-1和insulin的表達。當細胞擴增、融合至80-90％時，改變細胞培養(yǎng)方案，通過含有exendin-4、KGF、NIC等細胞因子和葡萄糖的誘導分化培養(yǎng)基，對胰腺導管上皮細胞進行向胰島細胞團轉分化的實驗。(2)觀察細胞生長狀態(tài)的變化并攝片。4周后對轉分化后的細胞進行鑒定：DTZ染色；RT-PCR檢測Glut-2、PDX-1、insulin、glucagon基因的表達；行葡萄糖刺激試驗，用RIA法檢測其胰島素和C肽的分泌功能。(3)分離純化大鼠天然胰島，用同樣的方法檢測胰島素和C肽的分泌功能，并與誘導轉分化所得細胞團進行比較。 結果：(1)成功分離、純化了大鼠胰腺導管上皮細胞，并使其得到有效擴增，有效抑制了胰島細胞和成纖維細胞的污染，，純化后大部分細胞表達ck-19(胰腺導管上皮細胞的標志)，而RT-PCR顯示PDX-1、insulin表達陰性。(2)誘導后細胞逐漸增大、變形，并出現類圓形小細胞，聚集成團，繼而得到圓形或橢圓形的胰島樣團狀結構，細胞團慢慢增大、增多，4周后細胞團DTz染色陽性，RT-PCR顯示Glut-2、PDX-1、insulin、glucagon表達陽性。(3)對培養(yǎng)液上清的檢測，誘導轉分化所得胰島細胞團在高糖刺激下的胰島素分泌量為低糖時的6倍，在低濃度(3.3mM)和高濃度(16.7mM)時，誘導后胰島細胞團胰島素的分泌量分別約為天然胰島的1／30和1／35。C肽的分泌量亦可隨葡萄糖濃度的升高而增加，在低濃度和高濃度時分別為天然胰島的1／32和1／26。 結論：大鼠胰腺導管上皮細胞在體外可得到有效擴增并具有干細胞潛能，在細胞因子的作用下，可以進一步轉分化為胰島樣細胞團，該胰島樣細胞團可隨葡萄糖濃度的高低調節(jié)胰島素和C肽的分泌。但與天然胰島相比，胰島素和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.
【學位授予單位】：暨南大學
【學位級別】：碩士
【學位授予年份】：2006
【分類號】：R329

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