Nanog和C-sis基因修饰骨髓间充质干细胞自体移植治疗暴发性肝衰竭的实验研究

At the present, it is very difficult to treat fulminant hepatic failure with high mortality. Because of their pluripotent and self-renewing capacity, embryonic stem cells can be applied to treating fulminant hepatic failure. This application, however, is limited by ethics and immune responses triggered by allograft transplantation of embryonic stem cells. Nanog gene can effectively transform adult stem cells into embryonic-like stem cells. In this project, the expression plasmids(pcDNA3.1/Nanog, pcDNA3.1/Nanog/Oct4 and pcDNA3.1/Nanog/Sox2) will be established and then transfected into rat bone marrow-derived mesenchymal stem cells, which will bring about transforming these cells into embryonic-like stem cells. These embryonic-like stem cells are modified by pro-oncogene C-sis which can promote repairing tissue and eventually transplanted autologously into the damaged liver of rats. Through detecting and observing mortality of animal, biochemical indicators of blood, cell apoptosis, liver pathology as well as the specific markers of hepatocytes and bile duct cells, the repair effects of autologous transplantation of pure bone marrow-derived mesenchymal stem cells, pure embryonic-like stem cells, and embryonic-like stem cells modified by C-sis on the damaged liver will be evaluated and compared. Meanwhile, its mechanism will be studied too. This research could provide a new strategy for treating fulminant hepatic failure and the failure of other organs with embryo-like stem cells.

暴发性肝衰竭死亡率极高，治疗十分棘手。胚胎干细胞具有多潜能性和自我更新能力，可应用于暴发性肝衰竭的治疗。但伦理学问题和胚胎干细胞输入异体后所产生的免疫反应限制了其应用。Nanog基因能有效诱导成体干细胞转化为胚胎样干细胞。本项目将构建大鼠pcDNA3.1/Nanog、pcDNA3.1/Nanog/Oct4和pcDNA3.1/Nanog/Sox2表达质粒后转染大鼠骨髓间充质干细胞，诱导其转化为胚胎样干细胞。再用对组织修复有促进作用的原癌基因C-sis修饰胚胎样干细胞，最终自体移植入大鼠受损肝脏内。通过对动物死亡率、血生化指标、细胞凋亡、肝脏病理学以及肝细胞和胆管细胞特异性标志物的观察与检测，评估和比较单纯骨髓间充质干细胞、单纯胚胎样干细胞、C-sis修饰的胚胎样干细胞等自体移植对肝脏损伤的修复效果，并探讨其作用机制。本研究有望为胚胎样干细胞治疗暴发性肝衰竭乃至其它各类器官衰竭提供一条新途径。

暴发性肝衰竭（FHF）是由多种病因引起且突然出现的大量肝细胞坏死及严重肝功能损害，并在首发症状出现后8周内发生肝性脑病的一种综合征。原癌基因C-sis是血小板衍生生长因子B链的编码基因。很多研究表明，C-sis具有促进细胞增殖并抑制凋亡，进而促进组织修复的功能。本项目首先克隆了大鼠C-sis基因并构建了相应真核表达载体。从细胞水平，用H2O2诱导正常大鼠肝细胞BRL细胞凋亡，同时用重组质粒pcDNA3.1/C-sis转染细胞，最终检测细胞的增殖和凋亡；其次从动物水平，用LPS/D-GalN处理大鼠建立FHF模型，同时将重组质粒pcDNA3.1/C-sis注入大鼠体内进而进入肝脏，最终检测大鼠肝脏的增殖和凋亡，并计算实验大鼠死亡率。本研究结果显示，从离体细胞水平来看，H2O2能诱导正常大鼠肝细胞凋亡，并抑制其增殖，而C-sis基因表达上调能抑制H2O2的上述作用。即证实了C-sis基因能抑制受损肝细胞的凋亡，并促进其增殖。从在体动物水平来看，LPS/D-GalN能诱导大鼠发生FHF，而C-sis基因表达上调能减弱FHF的肝损伤程度，并降低其死亡率。即证实了C-sis基因能抑制FHF的肝损伤。本项目还克隆了大鼠Nanog基因并构建了相应的真核表达载体，将用于后续利用Nanog基因将骨髓间充质干细胞诱导为胚胎干细胞，并用于治疗FHF的实验。综上所述，本研究从动物和细胞水平均证实了，C-sis基因能抑制肝细胞的凋亡并减弱FHF的肝损伤。因此，C-sis可能成为FHF治疗的分子作用靶点，为治疗FHF提供新的可能途径。并且，本项目将为包括FHF在内的肝脏损伤的基因治疗提供理论与实践基础。已发表由本项目资助的论文共4篇，另有2篇已录用待发表，相应的SCI论文正在投稿中。

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