【摘要】： 目的研究成人骨髓間充質(zhì)干細(xì)胞(MSCs)的體外分離培養(yǎng)及向胰島素分泌細(xì)胞的定向誘導(dǎo)分化和鑒定方法,為糖尿病的治療提供種子細(xì)胞來(lái)源。方法1.分別采用全骨髓培養(yǎng)法與Percoll密度梯度離心法從成人骨髓中分離MSCs,相同條件下體外培養(yǎng),比較兩種方法所獲得的貼壁細(xì)胞克隆數(shù)、細(xì)胞形態(tài)、細(xì)胞表面標(biāo)志及向脂肪細(xì)胞的分化情況。2.將全骨髓培養(yǎng)法獲得的MSCs體外培養(yǎng)傳代,取3～5代細(xì)胞先后予以2-巰基乙醇、谷氨酰胺、表皮生長(zhǎng)因子(EGF)、堿性成纖維細(xì)胞生長(zhǎng)因子(bFGF)及尼克酰胺等,通過(guò)三階段向胰島樣細(xì)胞定向誘導(dǎo)分化。3.相差顯微鏡下觀察MSCs在誘導(dǎo)前后的形態(tài)變化、免疫熒光鑒定胰十二指腸同源異型盒基因(PDX-1)的表達(dá)、雙硫腙染色鑒定胰島β樣細(xì)胞團(tuán)、電化學(xué)發(fā)光法測(cè)定胰島素的分泌量。結(jié)果1.全骨髓培養(yǎng)法獲得的貼壁細(xì)胞克隆數(shù)明顯多于Percoll密度梯度離心法(P＜0.05)。兩種方法獲得的人MSCs形態(tài)無(wú)明顯差異,CD44陽(yáng)性表達(dá)率和CD34陰性表達(dá)率差異無(wú)統(tǒng)計(jì)學(xué)意義(P＞0.05),經(jīng)地塞米松、胰島素誘導(dǎo)后均可分化為脂肪細(xì)胞。2.MSCs經(jīng)2-巰基乙醇、尼克酰胺等誘導(dǎo)后,細(xì)胞逐漸變圓,誘導(dǎo)二階段細(xì)胞表達(dá)PDX-1,誘導(dǎo)三階段形成胰島樣細(xì)胞團(tuán),雙硫腙染色陽(yáng)性,葡萄糖刺激有胰島素釋放;而未經(jīng)誘導(dǎo)的MSCs呈長(zhǎng)梭形貼壁生長(zhǎng),無(wú)PDX-1表達(dá),雙硫腙染色陰性,無(wú)胰島素釋放。結(jié)論1.與Percoll密度梯度離心法比較,全骨髓培養(yǎng)法是更為簡(jiǎn)便、實(shí)用的MSCs體外分離方法之一。2.2-巰基乙醇、谷氨酰胺、EGF、bFGF及尼克酰胺等可在體外誘導(dǎo)成人MSCs轉(zhuǎn)分化為胰島素分泌細(xì)胞。
[Abstract]:Objective to study the methods of isolation and culture of adult bone marrow mesenchymal stem cells (MSCs) in vitro and their differentiation into insulin secreting cells in order to provide seed cells for the treatment of diabetes mellitus. Method 1. Whole bone marrow culture method and Percoll density gradient centrifugation method were used to isolate MSCs, from adult bone marrow in vitro. The number and morphology of adherent cells obtained by the two methods were compared. Cell surface markers and differentiation into adipocytes. The MSCs obtained by whole bone marrow culture was subcultured in vitro. The cells of 5 passages were treated with 2-mercaptoethanol, glutamine, epidermal growth factor (EGF),) basic fibroblast growth factor (bFGF) and nicotinamide, respectively. The differentiation of islet like cells was induced by three stages. The morphologic changes of MSCs before and after induction were observed under phase contrast microscope, the expression of PDX-1 in pancreaticoduodenal homologous box (PDX-1) was detected by immunofluorescence, the 尾 -like cell cluster of pancreatic islet was identified by dithizone staining, and the secretion of insulin was determined by electrochemiluminescence. Result 1. The number of adherent cells obtained by whole bone marrow culture was significantly higher than that by Percoll density gradient centrifugation (P < 0. 05). There was no significant difference in positive expression rate of CD44 and negative expression rate of CD34 between the two methods (P > 0. 05). After induced by dexamethasone and insulin, human MSCs could differentiate into adipocytes. 2. MSCs were differentiated into adipocytes by 2-mercaptoethanol. After the induction of nicotinamide, the cells gradually became round, PDX-1, was induced to form islet like cell mass in three stages, dithizone staining was positive, glucose stimulated insulin release, while the uninduced MSCs was fusiform adherent growth. No PDX-1 expression, no dithizone staining, no insulin release. Conclusion 1. Compared with Percoll density gradient centrifugation, the whole bone marrow culture method is more convenient. One of the practical methods of isolation of MSCs in vitro. 2.2-mercaptoethanol, Glutamine EGFN bFGF and nicotinamide can induce adult MSCs to differentiate into insulin-producing cells in vitro.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類(lèi)號(hào)】：R329

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