脐血间充质细胞诱导分化成肝细胞及治疗肝损伤的研究

发布时间：2018-04-28 20:32

本文选题：脐带血 + 间充质干细胞　；参考：《广西医科大学》2009年硕士论文

【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),在特定环境下诱导成肝样细胞并进行鉴别,观察MSCs及肝样细胞对大鼠慢性肝损伤的修复作用,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;流式细胞仪检测MSCs表面抗原CD34、CD44表达情况;用肝细胞生长因子(hepatocyte growth factor,HGF)及成纤维细胞生长因子-4(Fibroblast growthfactor-4)联合诱导P3代的MSCs成肝样细胞;采用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表达;采用透射电镜观察MSCs和诱导的肝样细胞的超微结构;将MSCs及诱导后的肝样细胞输注入慢性肝损伤的大鼠体内,取肝脏病理,观察两者对肝损伤的修复情况。结果1.接种的MSCs于48～72h后贴壁,巢样克隆,梭形,折光性强,3天后梭形细胞体积开始增大,15天后出现梭形细胞集落,呈平行或火焰状排列或漩涡状生长,三周后细胞呈80～90%融合;细胞传至第三代时,呈较均一的长梭形,形态基本无变化。2.经过传代、纯化、诱导的细胞经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.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为c D44+/c D34-的细胞占(98.59±4.53)%。5.移植了人MSCs及诱导后的肝样细胞的大鼠肝损伤病理轻于无治疗组,肝样细胞组的病理损伤轻于MSCs组。结论1.使用密度梯度离心法和贴壁法相结合可以从人脐带血中成功分离培养出MSCs,经过反复换液及传代培养后可获得纯度较高的MSCs。2.HGF+FGF-4能很好地诱导MSCs成为肝样细胞。3.40%四氯化碳腹腔注射可以成功造出大鼠慢性肝损伤模型,稳定性及重现性好。4.MSCs和诱导后的肝样细胞均有修复肝损伤的作用,且诱导后的肝样细胞优于MSCs。5.MSCs能通过冻存的方法长期保存。
[Abstract]:Objective to isolate umbilical cord blood from healthy parturient in vitro, culture mesenchymal stem cells, induce hepatoid cells in a specific environment and differentiate them, and observe the repair effect of MSCs and hepatoid cells on chronic liver injury in rats. It provides a new cell source for hepatocyte transplantation and bioartificial liver transplantation in end-stage liver disease. Methods umbilical cord blood was collected from normal parturient, isolated by density gradient centrifugation and adherent method, cultured and purified, and the expression of MSCs surface antigen CD34 and CD44 was detected by flow cytometry. Hepatoblast like cells of P3 generation were induced by hepatocyte growth factor (HGF) and fibroblast growth factor-4 (fibroblast growth factor-4), the expression of hepatocyte markers was detected by immunocytochemistry and glycogen expression was detected by glycogen staining. The ultrastructure of MSCs and induced hepatocytes were observed by transmission electron microscope (TEM), and the MSCs and induced hepatoid cells were injected into the rats with chronic liver injury to observe the repair of liver injury. Result 1. The MSCs inoculated at 48 ~ 72 h followed by adherent, nest-like clone, fusiform, and strong refraction. After 3 days, the fusiform cell volume began to increase. After 15 days, fusiform cell colonies appeared in parallel or flame-like arrangement or whirlpool growth. After 3 weeks, the cells were fused in 80% or 90%. When the cells passed to the third generation, they were more uniform and fusiform, with no change in shape. 2. After passage and purification, the induced cells gradually disappeared after 3-4 weeks, and the long fusiform cells became round or polygonal, with the appearance of granules in the cytoplasm and binuclear. 3. The detection of markers: in the non-induced group, the expression of Albium albium AlBN, glycogen, alpha feto protein (AFP) was not found at day 0, but no expression was found thereafter. In the induction group, the expression of AFPnALB and glycogen in hepatocytes could be detected. 4. The cell surface antigen (cD44 / cD34-) in the third passage was 98.59 卤4.53. The pathological changes of the transplanted MSCs and induced hepatoid cells were lighter than those of the untreated group, and the pathological damage of the hepatoid cells group was lighter than that of the MSCs group. Conclusion 1. Using density gradient centrifugation combined with adherent method, MSCs can be isolated and cultured from human umbilical cord blood successfully. After repeated fluid exchange and subculture, high purity MSCs.2.HGF FGF-4 can be obtained to induce MSCs into hepatoid cells. 3.40% tetrachlor. The model of chronic liver injury in rats can be established successfully by intraperitoneal injection of carbon. Good stability and reproducibility. 4. MSCs and induced hepatoid cells can repair liver injury, and the induced hepatoid cells are better than MSCs.5.MSCs through cryopreservation for a long time.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329;R575

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