骨髓间充质干细胞分化为肝样细胞治疗HBV小鼠肝损伤的实验研究

发布时间：2018-04-22 08:21

本文选题：转基因HBV小鼠 + 肝损伤　；参考：《新疆农业大学》2012年硕士论文

【摘要】：研究背景与目的：肝脏疾病是影响人类健康的最为常见疾病之一。各种有害因素导致的肝损伤主要有病毒性肝炎、酒精性肝炎和药物性肝炎等类型，其临床表现为肝坏死、脂肪肝、胆汁淤积、肝纤维化、肝硬化以及肝癌等。目前对肝损伤的治疗仍是一个全球性的严峻课题，而干细胞移植用于治疗肝损伤是一种新方法并显示出良好的应用前景。骨髓间充质干细胞具有极强的多向分化潜能和自我复制能力,有望成为治疗肝损伤的一种新型种子细胞。本课题基于上述问题,采用HGF和EGF联合诱导BMSCs21天为肝样细胞，再将肝样细胞移植到肝损伤的HBV小鼠肝脏来治疗，并通过鉴定来确定细胞移植治疗肝损伤的效果，以期为临床研究提供实验基础。方法：(1)分别建立HBV转基因小鼠和C57BL/6小鼠骨髓间充质干细胞BMSCs的分离、培养体系，并进行比较研究。(2)将纯化后的BMSCs在肝样细胞生长因子HGF和表皮生长因子EGF的作用下进行诱导培养后,免疫细胞化学检测肝样细胞表面标志物角蛋白CK18及糖原PAS的表达,明确是否可分化为肝样细胞（3）制作小鼠肝损伤模型，，将标记后的BMSCs移植入肝损伤模型小鼠体内,荧光显微镜检测干细胞在肝内的定植情况,检测血清肝功能及免疫组化检测肝组织角蛋白的表达情况。结果：(1) C57BL/6小鼠的原代BMSCs生长迅速，按1:3传代后呈漩涡状生长，形态类似成纤维细胞。HBV转基因小鼠原代BMSCs生长缓慢；以1:2传代后生长缓慢，并且有明显的血清依赖性及密度依赖性。(2) BMSCs经HGF和EGF诱导培养后,免疫细胞化学检测肝样细胞表面标志物角蛋白CK18及糖原PAS的表达均为阳性。(3)血清ALT及AST活性结果，模型组A2与正常对照A1组存在显著差异（P0.05）；模型组A2与干预组B1、B2在第四周有显著差异（P0.05）；干预组B1、B2在第四周有显著差异（P0.05）。结论：(1)与C57BL/6小鼠BMSCs比较，HBV转基因小鼠BMSCs体外生长存在显著的血清依赖性及密度依赖性。(2) BMSCs经HGF和EGF诱导培养后,可分化为肝样细胞。(3)在第四周时两个干预组BMSCs组、BMSCs诱导为肝样细胞组之间也存在显著差异，从ALT及AST活性及荧光标记结果来看，BMSCs诱导为肝样细胞组对受损肝脏的功能恢复上有更好的作用。
[Abstract]:Background and objective: liver disease is one of the most common diseases affecting human health. The main types of liver injury caused by various harmful factors are viral hepatitis, alcoholic hepatitis and drug hepatitis. Their clinical manifestations are liver necrosis, fatty liver, cholestasis, liver fibrosis, liver cirrhosis and liver cancer. At present, the treatment of liver injury is still a severe global problem, and stem cell transplantation is a new method for the treatment of liver injury and has shown a good application prospect. Bone marrow mesenchymal stem cells (BMSCs) have strong multidirectional differentiation potential and self-replicating ability and are expected to be a new type of seed cells for the treatment of liver injury. Based on the above problems, we used HGF and EGF to induce BMSCs21 cells to be hepatoid cells, then transplanted hepatoid cells into the liver of HBV mice with liver injury, and confirmed the effect of cell transplantation on liver injury by identification. In order to provide experimental basis for clinical research. Methods the isolation and culture system of BMSCs from bone marrow mesenchymal stem cells of HBV transgenic mice and C57BL/6 mice were established. The purified BMSCs was induced by hepatocyte growth factor (HGF) and epidermal growth factor (EGF). The expression of keratin CK18 and glycogen PAS was detected by immunocytochemistry. To determine whether it is possible to differentiate into hepatocyte-like cells, and to make the liver injury model in mice, the labeled BMSCs was transplanted into the liver injury model mice, and the colonization of stem cells in the liver was detected by fluorescence microscope. Serum liver function and immunohistochemistry were used to detect the expression of keratin in liver tissue. Results the primary BMSCs of C57BL/6 mice grew rapidly, and grew in whirlpool form after subculture at 1:3. The primary BMSCs of HBV-like fibroblasts grew slowly, but at 1:2, it grew slowly. The expression of keratin CK18 and glycogen PAS on the surface of hepatoid cells was detected by immunocytochemistry. The results of serum ALT and AST activity were also observed. There were significant differences between model group A2 and normal control group A1 (P 0.05), model group A 2 and intervention group B 1 B 2 in the fourth week (P 0.05), and intervention group B 1 B 2 had significant difference in the fourth week (P 0.05). Conclusion compared with BMSCs of C57BL/6 mice, the growth of BMSCs in BMSCs transgenic mice is significantly serum-dependent and density-dependent. 2) BMSCs was induced by HGF and EGF. At the fourth week, there was also significant difference between the two intervention groups in inducing BMSCs into hepatoid cells. From the results of ALT and AST activity and fluorescence labeling, it was found that ALT induced by hepatocyte-like cells had a better effect on the recovery of damaged liver function.
【学位授予单位】：新疆农业大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R-332;R575

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