基于骨髓间充质干细胞“归巢”的补肾活血化浊方调控Wilson病TX小鼠肝纤维化肝再生代谢组学研究

Liver failure induced by hepatic fibrosis is the main cause of death in patients with Wilson disease,it is the difficulty point of encephalopathy and liver disease.The study group carried on the related research in early stage to WD hepatic fibrosis,found that Gandouling can interfere with the expression of p38MAPK signaling pathway and induced the apoptosis of hepatic stellate cells ,and it has obvious regulation effect on WD marker metabolites.BMSCs is a new methods in the study of hepatic fibrosis,liver regeneration is a new target for prevention and treatment of hepatic fibrosis.However, it is not clear that the mechanism of Gandouling promoting the BMSCs to intervence liver regeneration .According to the pathogenesis of kidney essence deficiency, phlegm and blood stasis blocking collaterals of WD hepatic fibrosis , and the TCM attribute of kidney essence of BMSCs, so we think,tonifying kidney and promoting blood circulation and removing turbidity, which is not only consistent with the TCM pathogenesis of WD hepatic fibrosis,but also agree with the TCM connotation of BMSCs,Gandouling can promote the homing of BMSCs, regulate the liver regeneration, treatment the hepatic fibrosis. This subject continue to take the p38MAPK signaling pathway as the breakthrough point, metabonomics as the research tool,combining traditional Chinese medicine research with stem cell technology,seeking for the target of combining traditional Chinese medicine compound with stem cells,to find a new therapeutic method for the difficult disease of hepatic fibrosis in WD.

Wilson病(Wilson disease,WD)肝纤维化引起的肝功能衰竭是WD患者死亡主要原因，是脑病与肝病学科交叉研究的难点。课题组前期对WD肝纤维化进行相关研究，发现肝豆灵有干预p38MAPK信号通路诱导肝星状细胞凋亡的作用，对WD代谢产物有调节效应。骨髓间充质干细胞(BMSCs)是肝纤维化研究的新手段，肝再生是肝纤维化防治的新靶点，但肝豆灵促进BMSCs干预WD肝纤维化、调控肝再生机制尚不明确。根据WD肝纤维化“肾精亏虚、痰瘀阻络”病机及BMSCs中医“肾精”属性，课题组假设：补肾活血化浊法既符合WD肝纤维化中医病机，同时与BMSCs中医内涵相切合，肝豆灵能够促进BMSCs归巢，调控肝再生，治疗肝纤维化。本课题继续以p38MAPK信号通路为切入点，代谢组学为研究工具，将中医药基础研究与干细胞技术有机结合，寻找中药复方与干细胞结合的靶点，为疑难疾病WD肝纤维化寻求新的治疗方法。

Wilson病(Wilson disease,WD)肝纤维化引起的肝功能衰竭是WD患者死亡主要原因，骨髓间充质干细胞(BMSCs)是肝纤维化研究的新手段，肝再生是肝纤维化防治的新靶点，本课题探讨补肾活血化浊方肝豆灵促进BMSCs归巢，调控肝再生治疗肝纤维化的机制。项目研究内容及结果如下：.①采用全骨髓冲洗法分离小鼠骨髓间充质干细胞，进行形态学观察，免疫荧光染色鉴定 BMSCs，LV3­空载慢病毒标记 BMSCs、鉴定，说明成功分离培养BMSCs；流式细胞仪检测LV3­空载慢病毒标记的 BMSCs表面抗原，说明慢病毒感染不改变BMSCs的表型，可用于下一步BMSCs移植。.②LV3­空载慢病毒标记BMSCs在肝脏表达，移植后第4周，激光共聚焦显微镜显示肝脏切片可见绿色荧光表达，可证明LV3-空载慢病毒标记的BMSCs存活并定植于小鼠肝脏，表明标记成功。.③HE、Mosson染色及TX小鼠肝组织羟脯氨酸(Hyp)含量表达，说明4月龄TX小鼠符合肝纤维化模型要求，证实BMSCs及肝豆灵可改善TX小鼠肝纤维化程度。.④TUNEL法检测肝状细胞凋亡，说明补肾活血化浊方联合BMSCs移植治疗可有效抑制肝细胞凋亡修复肝损伤，改善肝纤维化程度。.⑤采利用Western-Blot及RT-PCR技术检测TGF-β1，BMP-7，p38MAPK，p-p38MAPK的表达，分析补肾活血化浊方肝豆灵及BMSCs均能通过降低TGF-β1，p38MAPK，p-p38MAPK的表达，提高BMP-7的表达，同时肝豆灵具有促进BMSCs归巢，改善肝纤维化的作用。.⑥利用代谢组学技术对TX小鼠肝纤维化肝组织相关代谢物水平进行检测标记，从代谢组学角度分析补肾活血化浊方促进BMSCs改善Wilson病肝纤维化的作用机制。不同干预方法可能通过调节小分子物质代谢紊乱，对TX小鼠肝纤维化有一定的改善作用。.本课题按照申请书，以实验动物为研究对象，环环相扣，验证了预定设想，完成了预期计划，为肝豆状核变性的治疗难点肝纤维化探索新的治疗手段，为中药复方制剂肝豆灵临床应用提供实验基础及理论依据。