本文關(guān)鍵詞： 胰腺導(dǎo)管干細(xì)胞 轉(zhuǎn)分化 胰島內(nèi)分泌細(xì)胞 天然胰島 基因表達(dá)譜　出處：《暨南大學(xué)》2006年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：體外分離培養(yǎng)大鼠胰腺導(dǎo)管干細(xì)胞，并對(duì)其純化、擴(kuò)增、鑒定，轉(zhuǎn)分化為胰島內(nèi)分泌細(xì)胞，分離純化大鼠天然胰島，通過(guò)基因表達(dá)譜芯片研究轉(zhuǎn)分化的胰島內(nèi)分泌細(xì)胞與天然胰島細(xì)胞在成熟度上的差異，探討哪些基因?qū)D(zhuǎn)分化起著關(guān)鍵性調(diào)節(jié)作用。 方法：酶消化法分離大鼠胰腺導(dǎo)管干細(xì)胞，CK-19、PDX-1進(jìn)行免疫細(xì)胞化學(xué)染色鑒定。密度梯度離心法分離純化大鼠天然胰島，，雙硫腙染色鑒定轉(zhuǎn)分化的胰島內(nèi)分泌細(xì)胞與天然胰島細(xì)胞。對(duì)轉(zhuǎn)分化的胰島內(nèi)分泌細(xì)胞進(jìn)行葡萄糖刺激實(shí)驗(yàn)，ELISA法測(cè)定胰島細(xì)胞的胰島素分泌量，對(duì)誘導(dǎo)分化不同時(shí)間點(diǎn)的干細(xì)胞培養(yǎng)基進(jìn)行C肽測(cè)定。采用大鼠22000位點(diǎn)寡核苷酸芯片對(duì)轉(zhuǎn)分化的胰島內(nèi)分泌細(xì)胞和分離純化的天然胰島提取RNA樣品進(jìn)行3次雜交重復(fù)實(shí)驗(yàn)，篩選差異性表達(dá)的基因。 結(jié)果：分離培養(yǎng)的大鼠胰腺導(dǎo)管干細(xì)胞經(jīng)免疫組化染色呈CK-19、PDX-1染色陽(yáng)性。經(jīng)過(guò)誘導(dǎo)分化后，細(xì)胞形成類胰島樣細(xì)胞團(tuán)，呈巢團(tuán)樣聚集，雙硫腙染色為棕紅色。體外分離的大鼠天然胰島與轉(zhuǎn)分化的胰島內(nèi)分泌細(xì)胞的基因表達(dá)譜芯片差異性分析發(fā)現(xiàn)一些參與胰島發(fā)育的關(guān)鍵基因的表達(dá)明顯下調(diào)。 結(jié)論：體外培養(yǎng)條件下大鼠胰腺干細(xì)胞可增殖并轉(zhuǎn)分化為胰島樣細(xì)胞，轉(zhuǎn)分化的培養(yǎng)基中胰島素分泌水平遠(yuǎn)低于天然胰島細(xì)胞，表明轉(zhuǎn)分化而來(lái)的胰島內(nèi)分泌細(xì)胞功能與天然胰島仍存在較大差異�；虮磉_(dá)譜芯片研究發(fā)現(xiàn)轉(zhuǎn)分化的胰島內(nèi)分泌細(xì)胞與天然胰島細(xì)胞基因表達(dá)差異顯著，證明轉(zhuǎn)分化的胰島內(nèi)分泌細(xì)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.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：R329

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