【作者】 王伟；

【导师】 撒亚莲；

【作者基本信息】 昆明医科大学 ， 人体解剖与组织胚胎学， 2012， 硕士

【摘要】 目的：体外分离培养人脂肪源性干细胞(human adipose derived stem cells, hADSCs),观察hADSCs的细胞形态,检测hADSCs的生长曲线、表面标志物及其在体外的分化潜能,初步探讨Insulin、Nestin、CK19在hADSCs分化为胰岛素分泌样细胞过程中的表达。方法：采用Ⅰ型胶原酶消化法从成人皮下脂肪组织分离培养hADSCs,在倒置显微镜下观察hADSCs的形态特点和生长特征,用细胞计数法绘制hADSCs(P3、P8)的生长曲线,用免疫组织化学方法检测波形蛋白,流式细胞术分析细胞表面标志物CD34、CD45、CD44、CD90、CD105的表达以及观察hADSCs分化为脂肪样细胞、成骨样细胞的潜能,以及通过三步诱导法诱导hADSCs分化为胰岛素分泌样细胞。首先采用含2-巯基乙醇(2-mercaptoethanol,2-ME)、胎牛血清(fetal bovine serum, FBS)的H-DMEM培养基诱导6天,之后用含2-ME、碱性成纤维细胞生长因子(basic Fibroblast Growth Factor, bFGF)、表皮细胞生长因子(Epidermal Growth Factor, EGF)、B27、牛血清白蛋白(bovine serum albumin, BSA)的H-DMEM培养基诱导6天,最后用含2-ME、尼克酰胺、B27、BSA的H-DMEM培养基诱导6天。对照组细胞采用含10%FBS的H-DMEM培养基培养。观察细胞形态及生长特征,对诱导后细胞进行双硫腙染色以及免疫组化检测Insulin、 Nestin、CK19的表达。结果：在倒置相差显微镜下观察,新鲜分离的hADSCs在48-72小时贴壁,为成纤维样细胞。细胞传代后3天内处于潜伏期,第4天进入生长期,第7天进入平台期。细胞的增殖能力随传代次数的增加而有所下降,细胞培养至9代,生长基本停滞,呈老化状态。免疫组织化学方法检测hADSCs表达波形蛋白,流式细胞术检测第3代hADSCs表达CD34、CD45、CD44、CD90、CD105阳性率分别是1.5%、0.0%、100.0%、98.6%、99.5%。选用P3的hADSCs用于诱导分化试验hADSCs经定向诱导成脂分化后,胞质出现脂滴,油红O染色为阳性hADSCs经定向诱导成骨分化后,可见钙结节,茜素红染色为阳性。在诱导hADSCs分化为胰岛素分泌样细胞的第一阶段,在倒置显微镜下观察到细胞部分脱落,存活细胞的折光性增强,形态未发生明显改变,双硫腙染色为阴性,免疫组织化学方法检测Nestin、CK19和Insulin均为阴性表达。在诱导的第二阶段,细胞大量增殖,部分细胞形态由长梭形变为圆形,双硫腙染色阴性,免疫组化检测Nestin为阳性表达,而CK19和Insulin为阴性表达。在诱导的第三阶段,诱导细胞成团聚集,双硫腙染色阳性,免疫组化检测Nestin、CK19和Insulin均阴性表达。对照组染色均为阴性。结论：Ⅰ型胶原酶消化法可从成人皮下脂肪组织中分离获取hADSCs,其具有多向分化潜能；在hADSCs分化为胰岛素分泌样细胞的过程中有Nestin表达,双硫腙染色阳性。更多还原

【Abstract】 Objective:To explore a method for effective isolation and cultivation of hADSCs, observe morphological feature, growth kinetics, surface marker expressions as well as differentiation potential. To investigate expression of Insulin、Nestin、 CK19in the course of hADSCs differentiate into insulin-producing cells induced by multiple chemical regents in vitro.Methods:hADSCs were isolated and cultured from adult subcutaneously adipose tissue by collagenase type I. Growth curve of hADSCs in3rd and8th passage was measured by cells counting, and vimentin was detected by immunocytochemistry. The phenotypic analysis including CD34. CD45、CD44. CD90、CD105was tested by flow cytometry. hADSCs were observed differentiation potential towards the osteogenic and adipogenic lineages. The hADSCs in3rd~5th were induced towards insulin-producing cells through three developmental stages. Firstly, hADSCs were incubated in H-DMEM supplemented with2-mercaptoethanol and FBS for6days. hADSCs were then induced by H-DMEM containing bFGF、EGF、2-mercaptoethanol、B27and BSA for another6days. Finally, hADSCs were cultured in H-DMEM supplemented with nicotinamide、2-mercaptoethanol、B27and BSA for another6days. In the control group, hADSCs were incubated in H-DMEM. The morphological changes of hADSCs in different medium were observed under phase contrast inverted microscope,and the induced cells were detected by dithizone staining. At days6,12and18, the expression of Insulin、Nestin and CK19was tested by immunohistochemistry.Results:hADSCs appeared fibroblast-like, spindle shape and homogeneous arranged parallel under phase contrast inverted microscope. hADSCs were still in latent phase after being subculture for2~3days, followed by logarithmical proliferation from4 days, reached the growth platform at7days. Serial subcultivation of hADSCs, its growth became slow, and gradually appeared senescent. hADSCs could be subcultured until passage9. Immunohistochemical staining showed hADSCs in P3expressed vimentin. FCM results indicated that the positive rate of CD34、CD45、 CD44、GD90、CD105was1.5%、0.0%、100.0%、98.6%、99.5%, respectively. After adipocyte like cells committed induction, hADSCs changed to round and with lipid vacuoles accumulated in the cytoplasm displayed positivie staining with oild-red O. Calcium nodules could be seen after osteogenic induction and alizarin red S staining was positive. At the first stage of being induced towards insulin-producing cells, hADSCs had a good refractive capacity, whereas the number of cells was decreased. Both dithizone staining and immunohistochemistry testing of Nestin、Insulin、CK19were negative at6days. At12days, the induced cells gradually become round, Nestin positive was detected by immunohistochemistry, but Insulin and CK19were negative. Dithizone staining was no signal. After18days of induction, induced cells could be self-assemble to form clusters. Dithizone staining was positive, whereas Nestin、 Insulin and CK19was negative. In the control group, dithizone staining and immunohistochemistry detecting were no signal.Conclusion:hADSCs can be effectively isolated from adult subcutaneously adipose tissue with collagenase type I, which possess the multiple differentiation potential. During human adipose derived stem cells differentiate into insulin-producing cells, induced cells appeared Nestin positive tested by immunohistochemistry as well as Dithizone staining was shown to contain insulin-positive cells.更多还原