人脐带间充质干细胞在肝部分切除模型大鼠体内向肝细胞分化的研究

发布时间：2018-03-28 14:14

本文选题：间充质干细胞　切入点：肝细胞　出处：《暨南大学》2012年硕士论文

【摘要】：目的探讨人脐带间充质干细胞（mesenchymal stem cells，MSCs）移植到肝部分切除模型大鼠体内能否存活，并分化为肝细胞，通过相关的实验方法检测人脐带MSCs能否改善肝损伤大鼠的肝功能，进一步为人脐带MSCs运用于临床提供动物实验的研究依据。方法用胶原酶消化法从人的脐带中分离出干细胞，培养并检测干细胞的细胞表面标志。取第5代人脐带MSCs用PKH26标记。将72只SD大鼠随机分成三组，即实验组、模型组和对照组，每组24只。三组SD大鼠，用戊巴比妥腹腔注射麻醉。实验组：双重结扎大鼠肝脏膈叶的肝蒂,切除肝叶，并将PKH26染色液标记过的细胞悬液注入实验组大鼠的门静脉。模型组：按上述方法切除相同比例的肝脏，并予以门静脉注射等体积的生理盐水。对照组：仅翻动肝脏，不切肝，予以门静脉注射等体积的生理盐水。各组术后取背部皮下注射生理盐水30ml/kg，补充术中体液的丢失。各组于术后第3、9、15、21天，随机取6只大鼠，开腹取出大鼠肝脏，观察人脐带MSCs能否存活,以及移植细胞能否向肝细胞分化,能否表达肝细胞的标记物白蛋白。检测指标：1.大鼠血清谷丙转氨酶、谷草转氨酶的活性；2.大鼠血清胆汁酸、白蛋白；3.大鼠的肝脏指数；4.制作肝脏病理切片并对肝脏的病变程度进行病理评分。结果人脐带MSCs能在体外大量扩增，移植到肝部分切除模型大鼠肝脏后细胞能够存活，并表达肝细胞标记物白蛋白。PKH26标记后细胞在荧光显微镜下发红色荧光。荧光显微镜下可见肝脏冰冻切片中散在分布的标记细胞，免疫荧光染色大多数标记细胞白蛋白染色阳性，并发绿色荧光。对照组相关的实验检测指标无明显变化，与实验组和模型组比较有统计学意义（p0.05）。实验组和模型组比较，实验组的ALT、AST的检测值在第9天明显低于模型组，TBA的检测值在第21天明显低于模型组，ALB检测值在第15天明显高于模型组，以上时间点两组比较有统计学意义（p0.05）。结论 1.人脐带间充质干细胞在体外可以成功分离并且培养，，其具有很强的增殖、分化潜能。 2.人脐带间充质干细胞移植至大鼠体内能够存活并分化为肝细胞,可改善肝损伤大鼠的肝功能，人脐带间充质干细胞可作为肝脏细胞移植的重要来源。
[Abstract]:Purpose. To investigate whether human umbilical cord mesenchymal stem cells can survive and differentiate into hepatocytes after partial hepatectomy in rats, and to investigate whether human umbilical cord MSCs can improve liver function in rats with liver injury. Further more, the application of human umbilical cord MSCs in clinical research provides evidence for animal experiments. Method. Stem cells were isolated from human umbilical cord by collagenase digestion, and the surface markers of stem cells were cultured and detected. The fifth passage of human umbilical cord MSCs was labeled with PKH26. 72 SD rats were randomly divided into three groups: experimental group, model group and control group. 24 rats in each group were anesthetized by intraperitoneal injection of pentobarbital. Experimental group: double ligation of liver pedicle of the phrenic lobe of rat liver, resection of liver lobe, The cell suspension labeled with PKH26 staining solution was injected into the portal vein of the experimental group. The model group: the liver was resected in the same proportion according to the above method, and the same volume of normal saline was injected into the portal vein. In the control group, only the liver was turned over, but the liver was not cut off. The rats in each group were injected with saline of the same volume by portal vein, 30 ml / kg of normal saline was injected subcutaneously into the back of each group after operation, and the body fluid was lost during rehydration. Six rats were randomly selected from each group on the 3rd day, 1521 days after operation, and the liver was taken out by laparotomy. To observe whether human umbilical cord MSCs can survive, whether the transplanted cells can differentiate into hepatocytes and whether they can express albumin, the marker of hepatocytes. Detection index: 1. The activity of serum alanine aminotransferase (alt) and aspartate aminotransferase (alt) in rat serum. Albumin 3.The liver index of rats was 4.The pathological sections of the liver were made and the pathological grade of the liver was evaluated. Results. Human umbilical cord MSCs can be expanded in vitro, and the cells can survive after transplantation to the liver of partially resected rat liver. The labeled hepatocyte albumin. PKH26 labeled hepatocytes showed red fluorescence under fluorescence microscope. The distribution of labeled cells in frozen sections of liver was observed under fluorescence microscope, and most of the labeled cells were positive for albumin staining by immunofluorescence staining. Compared with the experimental group and the model group, there was significant difference between the experimental group and the model group, the experimental group and the model group, the experimental group and the model group, the experimental group and the model group, the experimental group and the model group were compared with each other. The detection value of alt AST in the experimental group was significantly lower than that in the model group on the 9th day and the detection value of TBA in the model group on the 21st day was significantly lower than that in the model group on the 15th day. The above time points were significantly higher in the two groups than in the model group. Conclusion. 1. Human umbilical cord mesenchymal stem cells were successfully isolated and cultured in vitro. 2.Human umbilical cord mesenchymal stem cells can survive and differentiate into hepatocytes, which can improve the liver function of injured rats. Human umbilical cord mesenchymal stem cells can be used as an important source of liver cell transplantation.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R657.3;R-332

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