肝大部切除鼠血清诱导骨髓干细胞向肝前体细胞的分化及其基因表达谱的变化

发布时间：2018-06-06 17:02

本文选题：肝大部切除鼠血清 + 骨髓干细胞　；参考：《复旦大学》2005年博士论文

【摘要】：近年来的研究提示,成体骨髓干细胞(adult bone marrow stem cell,ABMSC)能转分化为肝细胞。这一现象为临床上应用ABMSC移植治疗肝损伤和急慢性肝功能衰竭展示了一个可喜的前景。但是ABMSC向肝细胞分化的影响因素及分化的机制尚不十分清楚。我们课题组先前有关肝再生的研究资料和国内外有关研究进展提示,虽然肝细胞生长因子(hepatocyte growth factor,HGF)是启动肝细胞DNA合成的主要因子,但肝细咆的生长、分化和增殖要受到机体整体状态、以及肝外和肝内多种因子和某些激素等的调控,而肝大部切除术后的血清则可能包含全部刺激因子和营养成分。因此本课题应用肝大部切除鼠血清和/或HGF,进行ABMSC向肝细胞分化的体内外实验研究。研究目的 1.应用肝大部切除鼠血清和/或HGF在体外诱导同系ABMSC向肝细胞分化,以探讨ABMSC向肝细胞前体分化和扩增的体外培养方法和条件。 2.诱导分化至一定阶段的大鼠ABMSC经不同途径(静脉注射、肝内注射和脾内注射)回输到同系大鼠肝损伤动物模型体内,观察其在体内向肝细胞分化以及参与肝组织重建的情况。 3.运用基因芯片技术对肝大部切除鼠血清诱导ABMSC向肝细胞分化的基因表达谱进行筛选,分析发生表达变化的基因,以期了解与ABMSC向肝细胞分化相关的特异性基因及它们之间的相互关系,从基因水平揭示了ABMSC向肝细胞分化中的分子机制。研究方法通过肝大部切除手术,制备肝再生动物模型,在术后24小时收集肝大部切除鼠血清。分离骨髓细胞体外培养,应用肝大部切除鼠血清诱导分化,利用免疫组化,RT-PCR,Western Blot等技术鉴定分化细胞。将已分化至一定阶段的肝前体细胞用CM-DiI进行标记后,回输到肝大部切除的肝再生动物模型体内,通过免疫组化和激光共聚焦显微镜等方法观察其在体内重建肝组织的情况。利用基因
[Abstract]:Recent studies suggest that adult bone marrow stem cells can be transformed into hepatocytes. This phenomenon presents a promising prospect for the clinical application of ABMSC transplantation in the treatment of liver injury and acute and chronic liver failure. However, the factors influencing the differentiation of ABMSC into hepatocytes and the mechanism of differentiation are not well understood. Our previous research data on liver regeneration and research progress at home and abroad suggest that although hepatocyte growth factor (HGF) is the main factor to initiate the synthesis of hepatocyte DNA, the growth of liver cells can be observed. Differentiation and proliferation are regulated by the whole state of the body, various factors and some hormones in the extrahepatic and intrahepatic liver, while the serum after subtotal hepatectomy may contain all the stimulating factors and nutrients. Therefore, the in vitro and in vivo study of the differentiation of ABMSC into hepatocytes was carried out by using rat serum and / or HGF after subtotal hepatectomy. Hepatectomy rat serum and / or HGF were used to induce the differentiation of homologous ABMSC into hepatocytes in vitro, in order to explore the culture methods and conditions for the differentiation and expansion of ABMSC into hepatocyte precursors in vitro. 2. After induction and differentiation to a certain stage, ABMSC was infused into the same rat model of liver injury by different ways (intrahepatic injection, intrahepatic injection and intrasplenic injection). To observe its differentiation into hepatocytes in vivo and participate in the reconstruction of liver tissue. 3. 3. The gene expression profiles of hepatocyte differentiation induced by serum from subtotal hepatectomy rats were screened by gene chip technique, and the genes that changed in expression were analyzed. In order to understand the specific genes related to the differentiation of ABMSC into hepatocytes and their relationship, the molecular mechanism of differentiation of ABMSC into hepatocytes was revealed at the gene level. Animal models of liver regeneration were established and serum samples were collected at 24 hours after hepatectomy. Bone marrow cells were isolated and cultured in vitro. Differentiation was induced by rat serum after subtotal hepatectomy. The differentiated cells were identified by immunohistochemistry with RT-PCR- Western blot. The liver precursor cells which had differentiated to a certain stage were labeled with CM-DiI and then transferred back to the model of liver regeneration after subtotal hepatectomy. The reconstitution of liver tissue in vivo was observed by immunohistochemistry and confocal laser microscopy. Utilization gene
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R329

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