不同纯度胰岛所含干细胞转分化培养前、后PDX-1的表达研究

发布时间：2018-06-07 02:34

本文选题：胰岛 + PDX-1　；参考：《重庆医科大学》2008年硕士论文

【摘要】： 目的:利用干细胞和非干细胞生长对血清的不同要求,获取不同纯度的大鼠胰岛中所含的干细胞,探讨PDX-1转分化前后的表达差异。方法:将30只雄性SD大鼠随机平均分成3组,均采用胶原酶V型通过胰管对胰腺进行灌注,切取胰腺,消化、离心获得胰岛细胞沉淀物。A组:胰岛沉淀物不进行纯化;B组:胰岛沉淀物中加入体积分数为25 %的Ficoll-400液纯化;C组:胰岛沉淀物依次加入25%及11 %的Ficoll-400纯化。将不同纯度胰岛首先接种于含10%胎牛血清Ham’s F-10培养液中连续培养36h;然后改用无血清Ham’s F-10培养液培养14天;最后以含KGF、BSA及ITS的无血清DMEM/F12(8mmol葡萄糖)培养液连续培养28天。在无血清Ham’s F-10培养液培养第14天以及含KGF、BSA及ITS的无血清DMEM/F12(8mmol葡萄糖)培养液培养第28天分别取部分贴壁细胞应用免疫细胞化学、RT-PCR方法分析所获得干细胞的胰十二指肠同源框(PDX-1)的表达。结果:(1)采用不同的纯化方法所得胰岛产量:A组:757.4±63.60个;B组:697.6±51.30个;C组:663.4±43.98个。B、C组间不具有统计学差异性(P㧐0.05),而A组与B、C组间有统计学差异性(P㩳0.05)。(2)采用不同的纯化方法所得胰岛纯度,A组:49.4±6.69%;B组:62.2±6.51%;C组:73.5±5.60%。各组间具有统计学差异性(P㩳0.05)。(3)免疫组化结果显示:各组细胞转分化培养前呈梭形或不规则形,胞浆PDX-1染色均呈阳性,面密度值:A组0.2331±0.0157,B组0.1143±0.0215,C组0.1051±0.0404,各组间有统计学差异性(P0.05);转分化培养后细胞呈卵圆形或不规则形且胞核PDX-1染色强阳性,面密度值:A组0.4207±0.1541,B组0.4383±0.1358,C组0.4201±0.1554,各组间没有统计学差异性(P0.05)(4)RT-PCR结果显示转分化培养前PDX-1 mRNA的表达(IOD值):A组:0.6405±0.1486,B组:0.4354±0.1422,C组:0.3011±0.1604,A组细胞转分化培养前PDX-1 mRNA表达较B、C两组强,各组间具有统计学差异性(P㩳0.05)。(5)RT-PCR结果显示转分化培养后PDX-1 mRNA的表达(IOD值),A组:0.7828±0.1341,B组:0.7583±0.1477,C组:0.7458±0.1509,各组间没有显著差异性(P0.05)。结论:(1)用三种不同纯化方法可制备不同纯度的胰岛,不同纯度胰岛中有干细胞存在。(2)胰腺干细胞可以在无血清条件下存活。(3)在未添加生长因子的条件下,胰腺干细胞可表现一定的增殖性。(4)不同纯度携带干细胞胰岛转分化培养前PDX-1表达有差别,纯度越低,表达越明显,转分化培养后PDX-1表达无差别;转分化后培养后PDX-1较转分化培养前表达强。(5)不同纯度胰岛所携带的干细胞经转分化培养后的子代细胞仍然保持干细胞的特征;KGF可能具有促进PDX-1表达的作用。
[Abstract]:Aim: to obtain stem cells from rat islets of different purity by using the different requirements of stem cell and non-stem cell growth on serum, and to explore the difference of expression of PDX-1 before and after transdifferentiation. Methods: thirty male Sprague-Dawley rats were randomly divided into three groups. The pancreas was perfused with collagenase V type through the pancreatic duct, the pancreas was removed and digested. Group A: islet precipitate was not purified in group B: group C was purified by adding 25% Ficoll-400 solution in islet precipitate, 25% and 11% Ficoll-400 were added in islet precipitate, respectively. The islets of different purity were first cultured in Ham's F-10 medium containing 10% fetal bovine serum for 36h, then cultured in serum-free Ham's F-10 medium for 14 days, and finally cultured in serum-free DMEM/F12(8mmol glucose medium containing ITS for 28 days. On the 14th day of serum-free Ham's F-10 culture and on the 28th day of serum-free DMEM/F12(8mmol glucose culture with KGF BSA and ITS, some adherent cells were obtained and analyzed by immunocytochemistry RT-PCR. Expression of PDX-1). Results the islet yield was obtained by using different purification methods. The yield of pancreatic islets was obtained by using different purification methods in group B: 697.6 卤51.30 in group C: 663.4 卤43.98. There was no statistical difference between group C and group A, but there was statistical difference between group A and group B, respectively. Group A: 49.4 卤6.69; group B: 62.2 卤6.51; group C: 73.5 卤5.60; The results of immunohistochemistry showed that the cells in each group were fusiform or irregular before transdifferentiation, and the cytoplasm PDX-1 staining was positive. The surface density of group A was 0.2331 卤0.0157 and group B was 0.1143 卤0.02154.There was a statistical difference between the two groups (P 0.05), and the cells were oval or irregular and strongly positive for nuclear PDX-1 staining after transdifferentiation. There was no statistical difference between the two groups. The results of RT-PCR showed that the expression of PDX-1 mRNA was stronger than that of the control group before differentiation and culture. The expression of PDX-1 mRNA was stronger in group A than that in group B: 0. 4354 卤0. 1422 and group C: 0. 3011 卤0. 1604. The expression of PDX-1 mRNA was stronger than that in group B before differentiation and culture. The expression of PDX-1 mRNA was significantly higher in group B than that in group B and C before differentiation and culture, and the expression of PDX-1 mRNA in group B was significantly higher than that in group B (0. 0405 卤0. 1486B vs 0. 3011 卤0. 1604A, P < 0. 05). The results of RT-PCR showed that the expression of PDX-1 mRNA in group A was 0.7828 卤0.1341 and group B was 0.7583 卤0.1477 and C was 0.7458 卤0.1509. There was no significant difference between the two groups. ConclusionThe islets of different purity can be prepared by three different purification methods. Stem cells exist in different purity islets. The pancreatic stem cells can survive in serum-free condition. Pancreatic stem cells showed a certain proliferative activity. (4) there were differences in PDX-1 expression before pancreatic islet transdifferentiation culture with different purity of carrying stem cells. The lower the purity, the more obvious the expression of PDX-1 was, but there was no difference in PDX-1 expression after transdifferentiation culture. The expression of PDX-1 after transdifferentiation was stronger than that before transdifferentiation. The progenies of stem cells with different purity of islets still maintained the characteristics of stem cells. KGF might promote the expression of PDX-1.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

【共引文献】