脐带间充质干细胞诱导分化成肝样细胞的研究

发布时间：2018-03-14 00:43

本文选题：脐带　切入点：间充质干细胞　出处：《大连医科大学》2012年硕士论文　论文类型：学位论文

【摘要】：目的：通过分离人脐带间充质干细胞(umbilical cord mesenchymal stem cells，UCMSCs)，采用系列细胞因子联合诱导，探索高效可靠的体外诱导分化为肝样细胞的技术方法。方法：采用健康产妇脐带，用酶消化法分离、培养脐带间充质干细胞（UCMSC）；流式细胞仪检测细胞抗原CD13、CD44、CD105、CD34、CD45、HLA-DR表达情况，鉴定分离的细胞是否为UCMSCs；用肝细胞生长因子(hepatocyte growth factor，HGF)、成纤维细胞生长因子-4(Fibroblast growth factor-4FGF-4)及白细胞介素6（Interleukin-6IL-6）进行组合分组：A组：仅基础培养基，即为阴性对照组；B组：基础培养基+HGF+FGF-4；C组：基础培养基+HGF+IL-6；D组：基础培养基+HGF+FGF-4+IL-6诱导培养第3代（P3）的脐带MSCs，用免疫细胞化学法检测细胞标志物AFP、ALB的表达情况，比较各个诱导组诱导成肝样细胞的细胞比例。数据用均数±标准差（x±s）表示，，采用SPSS18.0统计分析软件包进行分析，用单因素方差分析对资料进行统计分析。结果： 1.UCMSC的细胞形态接种的UCMSC多于48小时后贴壁，5天后有短梭形细胞长出，后渐由短梭形变为细长扁平的长梭形，15天后出现梭形细胞集落，呈漩涡状或长条形生长，1月后细胞相互接触,并铺满整个培养皿底部。 2.UCMSC鉴定取培养至第三代（P3）的脐带间充质干细胞行流式细胞仪检测细胞表面抗原表达，反映间充质干细胞的表面抗原CD13、CD44、CD105表达阳性，表达率分别为99.0%、98.4%、98.4%，而造血干细胞标志物CD34、CD45、HLA-DR表达阴性，表达率分别为4.6%、9.48%、5.04%。 3.UCMSC的诱导分化 3.1诱导成肝样细胞的细胞形态取培养至第三代的UCMSCs加入各细胞因子组合向肝样细胞诱导，经3~4周呈放射状的细胞排列结构逐步消失，长梭形细胞变类圆形或多角形，存在离散现象，胞浆出现颗粒，部分出现双核。 3.2肝样细胞标志物表达对诱导出的肝样细胞行其标志物甲胎蛋白(AFP)及白蛋白（ALB）检测显示：A组始终没有见到肝样细胞的标志物白蛋白(ALB)、甲胎蛋白(AFP)的表达。而B、C、D组可以检测到肝细胞的标志物AFP、ALB的表达，AFP在诱导7天时，不同诱导组（B、C、D）比较，D组表达率最高；至15天时各组表达均减少，但D组仍最高；至诱导20天时各组表达均消失。而ALB在诱导7天、15天、20天都有强表达。在诱导第7天，B组表达率最高；诱导15天、20天， B、D组表达率高于C组。结论： 1.使用酶消化法可以从人脐带中成功分离、培养出UCMSC。 2.应用肝细胞生长因子(hepatocyte growth factor，HGF)、成纤维细胞生长因子-4(Fibroblast growth factor-4FGF-4)及白细胞介素6（Interleukin-6IL-6）联合诱导UCMSCs可高效、稳定培养出肝样细胞。
[Abstract]:Aim: to isolate human umbilical cord mesenchymal stem cells (cord mesenchymal stem cells), and to explore a highly efficient and reliable method for inducing and differentiating into hepatoid cells in vitro by a series of cytokines. Methods: UCM SCC was cultured from umbilical cord of healthy pregnant women by enzyme digestion, and the expression of HLA-DR was detected by flow cytometry (FCM), and the expression of HLA-DR was detected by flow cytometry (FCM), and the expression of HLA-DR was detected by flow cytometry (FCM), and the expression of HLA-DR was detected by flow cytometry (FCM). To identify whether the isolated cells were UCMSCs or not, to classify the isolated cells into two groups: group A, combined with hepatocyte growth factor-HGF, fibroblast growth factor-4FGF-4, and interleukin 6 Interleukin-6IL-6). That is, the negative control group B: basic culture medium HGF FGF-4C group: basic medium HGF IL-6D group: basic medium HGF FGF-4 IL-6 induces and culture the third passage P3 umbilical cord MSCs. The expression of the cell marker AFPALB was detected by immunocytochemistry. The ratio of cells induced into hepatoid cells in each induction group was compared. The data were expressed by mean 卤standard deviation (x 卤s), analyzed by SPSS18.0 statistical analysis software package, and analyzed by single factor variance analysis (ANOVA). Results:. 1. After more than 48 hours of UCMSC inoculation, short fusiform cells of UCMSC grew after 5 days, and then gradually changed from short fusiform to slender fusiform cells. After 15 days, fusiform cell colonies appeared. After January, the cells contact each other and cover the bottom of the petri dish. 2. UCMSC identified the umbilical cord mesenchymal stem cells (UCMSC) cultured to the third generation of P3. Flow cytometry was used to detect the expression of surface antigen of mesenchymal stem cells, which indicated that the surface antigen of mesenchymal stem cells was positive. The expression rates of HLA-DR were 99.0 and 98.4, respectively, while the hematopoietic stem cell markers CD34, CD45, and HLA-DR were negative, and the expression rates were 4.69.48 and 5.04, respectively. 3. Differentiation of UCMSC. 3.1 the cell morphology of hepatoid cells induced to the third passage was induced by the addition of various cytokines to hepatoid cells. After 3 ~ 4 weeks, the radial cell arrangement gradually disappeared, and the long fusiform cells became round or polygonal. There is a discrete phenomenon, the cytoplasm appears granules, part of the binuclear. 3.2 expression of Hepatoid Cell markers in Hepatoid cells detected by AFP) and Albumin (ALB), the markers of Hepatoid cells (ALB) and Albumin (ALB) were not found in the liver like cells in group B: a, but the markers of ALB, AFP and AFP were not found in group B: C ~ (2 +), while those in group B ~ (2) C ~ (2) C ~ (3 +). The expression of AFPnALB, a marker of hepatocyte, was detected after 7 days of induction. The highest expression rate was observed in group D and decreased at day 15, but the expression rate in group D was still the highest in group D, but the expression rate in group D was higher than that in group C (P < 0.05). The expression rate of ALB was higher in group B than in group C on day 7 and day 20 after induction, and the expression rate of group B was higher than that of group C on day 7, and the expression rate of group B was higher than that of group C on the 7th day of induction, and the expression rate of ALB in group D was higher than that in group C on the 7th day of induction. Conclusion:. 1. UCMSCS could be successfully isolated from human umbilical cord by enzyme digestion. 2. The combination of hepatocyte growth factor-hGF-1, fibroblast growth factor-4FGF-4 and interleukin-6 Interleukin-6IL-6 can induce UCMSCs efficiently and stably.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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