人脐带血间充质干细胞体外定向分化为肝样细胞的研究

发布时间：2018-01-08 12:17

本文关键词：人脐带血间充质干细胞体外定向分化为肝样细胞的研究　出处：《广西医科大学》2008年硕士论文　论文类型：学位论文

【摘要】： 目的将来源于健康产妇的脐带血进行体外分离、培养出间充质干细胞(mesenchymal stem cells,MSCS),诱导成肝样细胞并进行鉴别,探讨间充质干细胞体外培养和定向分化为肝样细胞的可行性,为终末期肝病的肝细胞移植治疗和生物型人工肝提供一种新的细胞来源。方法采集正常产妇脐带血,密度梯度离心法和贴壁法分离,培养纯化脐血MSCS,使用肝细胞生长因子(hepatocyte growth factor,HGF)诱导MSCS。采用RT-PCR和免疫细胞化学法检测肝样细胞标志物的表达情况并进行糖原染色检测细胞糖原的表达;采用透射电镜观察间充质干细胞和诱导肝样细胞的超微结构;流式细胞仪检测间充质干细胞表面抗原CD34,CD44表达情况。结果1.采用密度梯度离心法和贴壁法分离接种的MSCs形态大小均一透亮度好,24小时内贴壁,以后逐渐增多,呈克隆性生长,3天左右可见梭形、三角形细胞,10天左右达到对数生长期。细胞传至第三代时,细胞融合成单层,梭形突起变长,排列有明显方向性,集落呈旋涡状生长,台盼蓝染色细胞存活率＞95%。2.经过传代,纯化,诱导的细胞由长梭形慢慢开始变圆,呈放射状的细胞排列结构逐步消失,形态变短,变类圆形或多角形,胞浆出现颗粒,部分出现双核,折光性强,分裂相多。非诱导组仍保留梭型的细胞形态。3.标志物检测:非诱导组始终没有见到肝细胞的标志物白蛋白(albium,ALB)、糖原表达,甲胎蛋白(alpha fetoprotein,AFP)第0天有少量表达此后不再表达;诱导组可以检测到肝细胞的标志物AFP、ALB和糖原的明显的表达。4.培养至第三代的细胞表面抗原CD44阳性表达,不表达CD34。结论1.采用密度梯度离心法和贴壁培养法可以在体外成功分离培养出脐带血MSCS。2.经HGF诱导的细胞初步确定为肝样细胞。
[Abstract]:To come from healthy maternal umbilical cord blood were isolated, cultured mesenchymal stem cells (mesenchymal stem cells, MSCS), and induced to differentiate into hepatocyte like cells were identified on mesenchymal stem cells in vitro and differentiate into hepatocyte like cells is feasible, end-stage liver disease liver cell transplantation and bioartificial liver provides a new cell source.
Methods normal maternal umbilical cord blood by density gradient centrifugation and adherent culture, purification of MSCS in umbilical cord blood, the use of hepatocyte growth factor (hepatocyte growth factor, HGF MSCS.) induced by RT-PCR and immunocytochemical method to detect hepatocyte like cells marked expression of glycogen staining cells and expression of glycogen by transmission; electron microscopic observation of the ultrastructure of mesenchymal stem cells and induced hepatocyte like cells; detection of mesenchymal stem cell surface antigen CD34 by flow cytometry, the expression of CD44.
Results 1. by density gradient centrifugation and adhering to the MSCs form wall isolation with uniform brightness, within 24 hours after the adherent was gradually increased, the clonal growth of about 3 days, visible fusiform, triangle cells, about 10 days to reach the logarithmic growth phase. When cells spread to the third generation, cell fusion single spindle, elongate, arranged in a clear direction, the colony was swirling growth, trypan blue staining and cell survival rate was 95%.2. after passage, purification, induced cells from spindle slowly began to turn round, radial arrangement of cells structure gradually disappear, form shorter, variable type round or polygonal, cytoplasm granules, part of the nucleus, strong refraction, split phase. Non induced group still retain markers of cell morphology of.3. spindle type: non induced group has not seen the hepatocyte marker albumin (albium, ALB), the expression of glycogen, AFP Protein (alpha fetoprotein, AFP) was expressed on a small scale for zeroth days, and then no longer expressed. The expression of AFP, ALB and glycogen in hepatocyte markers could be detected in the induction group,.4. was positive in CD44 cells cultured on the third generation, and CD34. was not expressed.
Conclusion 1.. Cord blood MSCS.2. can be successfully isolated and cultured in vitro by density gradient centrifugation and adherent culture. The cells induced by HGF are initially identified as hepatocyte like cells.

【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

【参考文献】