本文選題：胰島 + PDX-1��； 參考：《重慶醫(yī)科大學》2008年碩士論文

【摘要】： 目的:利用干細胞和非干細胞生長對血清的不同要求,獲取不同純度的大鼠胰島中所含的干細胞,探討PDX-1轉分化前后的表達差異。 方法:將30只雄性SD大鼠隨機平均分成3組,均采用膠原酶V型通過胰管對胰腺進行灌注,切取胰腺,消化、離心獲得胰島細胞沉淀物。A組:胰島沉淀物不進行純化;B組:胰島沉淀物中加入體積分數(shù)為25 %的Ficoll-400液純化;C組:胰島沉淀物依次加入25%及11 %的Ficoll-400純化。將不同純度胰島首先接種于含10%胎牛血清Ham’s F-10培養(yǎng)液中連續(xù)培養(yǎng)36h;然后改用無血清Ham’s F-10培養(yǎng)液培養(yǎng)14天;最后以含KGF、BSA及ITS的無血清DMEM/F12(8mmol葡萄糖)培養(yǎng)液連續(xù)培養(yǎng)28天。在無血清Ham’s F-10培養(yǎng)液培養(yǎng)第14天以及含KGF、BSA及ITS的無血清DMEM/F12(8mmol葡萄糖)培養(yǎng)液培養(yǎng)第28天分別取部分貼壁細胞應用免疫細胞化學、RT-PCR方法分析所獲得干細胞的胰十二指腸同源框(PDX-1)的表達。 結果:(1)采用不同的純化方法所得胰島產(chǎn)量:A組:757.4±63.60個;B組:697.6±51.30個;C組:663.4±43.98個。B、C組間不具有統(tǒng)計學差異性(P㧐0.05),而A組與B、C組間有統(tǒng)計學差異性(P㩳0.05)。(2)采用不同的純化方法所得胰島純度,A組:49.4±6.69%;B組:62.2±6.51%;C組:73.5±5.60%。各組間具有統(tǒng)計學差異性(P㩳0.05)。(3)免疫組化結果顯示:各組細胞轉分化培養(yǎng)前呈梭形或不規(guī)則形,胞漿PDX-1染色均呈陽性,面密度值:A組0.2331±0.0157,B組0.1143±0.0215,C組0.1051±0.0404,各組間有統(tǒng)計學差異性(P0.05);轉分化培養(yǎng)后細胞呈卵圓形或不規(guī)則形且胞核PDX-1染色強陽性,面密度值:A組0.4207±0.1541,B組0.4383±0.1358,C組0.4201±0.1554,各組間沒有統(tǒng)計學差異性(P0.05)(4)RT-PCR結果顯示轉分化培養(yǎng)前PDX-1 mRNA的表達(IOD值):A組:0.6405±0.1486,B組:0.4354±0.1422,C組:0.3011±0.1604,A組細胞轉分化培養(yǎng)前PDX-1 mRNA表達較B、C兩組強,各組間具有統(tǒng)計學差異性(P㩳0.05)。(5)RT-PCR結果顯示轉分化培養(yǎng)后PDX-1 mRNA的表達(IOD值),A組:0.7828±0.1341,B組:0.7583±0.1477,C組:0.7458±0.1509,各組間沒有顯著差異性(P0.05)。 結論:(1)用三種不同純化方法可制備不同純度的胰島,不同純度胰島中有干細胞存在。(2)胰腺干細胞可以在無血清條件下存活。(3)在未添加生長因子的條件下,胰腺干細胞可表現(xiàn)一定的增殖性。(4)不同純度攜帶干細胞胰島轉分化培養(yǎng)前PDX-1表達有差別,純度越低,表達越明顯,轉分化培養(yǎng)后PDX-1表達無差別;轉分化后培養(yǎng)后PDX-1較轉分化培養(yǎng)前表達強。(5)不同純度胰島所攜帶的干細胞經(jīng)轉分化培養(yǎng)后的子代細胞仍然保持干細胞的特征;KGF可能具有促進PDX-1表達的作用。
[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 鹵51.30 in group C: 663.4 鹵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 鹵6.69; group B: 62.2 鹵6.51; group C: 73.5 鹵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 鹵0.0157 and group B was 0.1143 鹵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 鹵0. 1422 and group C: 0. 3011 鹵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 鹵0. 1486B vs 0. 3011 鹵0. 1604A, P < 0. 05). The results of RT-PCR showed that the expression of PDX-1 mRNA in group A was 0.7828 鹵0.1341 and group B was 0.7583 鹵0.1477 and C was 0.7458 鹵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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R329

【共引文獻】

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本文編號：1989340