暴发型肝衰竭pDCs-CTL/CD4+CD25+Treg免疫网络紊乱特征分析及解毒化瘀颗粒的干预效应

The clinical treatment of fulminant hepatic failure is difficult because of its urgent seizure and poor prognosis. The "Triple fighting" theory almost comprehensively clarified its pathogenesis that the first mechanism of apoptosis and necrosis of hepatocytes is immune injury thus the obstacle of negative feedback mechanism for regulation is key.Our researching group has got satisfactory curative effects of reducing fatality by using "Jieduhuayu Granules" in treating the hepatic failure in earlier stage of this reseach and also make a discussion on the mechanism of pharmacologic action from protecting the mitochondria of liver cells and promoting the regeneration of liver cells. We has reaveled the immune pathogenesis of " first strive "of the fulminant hepatic failure and the links of intervening effects of "Jieduhuayu Granules" through the way of immune response process of pDCs-CTL and negative feedback of cellular immunity of CD4+CD25+Treg by the experimental methods of making MHV-3 virus-induced model of fulminant hepatic failure, separation technology using flow cytometry,antigen peptide four polymer technology，qRT-PCR technology and Immunohistochemistry technology,etc.This subject provides the technical support for "Jieduhuayu Granules" in treating the hepatic failure and shows the scientific connotation and value of TCM therapy of "Jieduannizhuan" in treating the hepatic failure and make further improvement on the survival rate in patients with hepatic failure.

暴发型肝衰竭起病急，预后差，临床治疗难度大。"三重打击"学说较全面地阐明了其发病机制，免疫损伤是肝细胞凋亡和坏死的首发机制，负反馈调节机制障碍是免疫调控网络紊乱的关键所在。本课题组前期应用解毒化瘀颗粒治疗肝衰竭，获得了降低病死率的满意疗效，并从肝细胞线粒体保护及促进肝细胞再生等方面探讨了作用机制。因此，本研究以MHV-3病毒诱导暴发型肝衰竭模型，运用流式细胞分选技术、抗原肽四聚体技术、qRT-PCR技术、免疫组化技术等实验方法,从pDCs-CTL免疫应答过程，CD4+CD25+Treg细胞免疫负反馈调节角度，揭示暴发型肝衰竭"首次打击"的免疫网络紊乱特征及解毒化瘀颗粒的干预效应环节。旨在为解毒化瘀颗粒治疗肝衰竭提供技术支撑；同时也为充分展示肝衰竭中医"截断逆转"法治疗理念的科学内涵及价值所系；为进一步提高肝衰竭患者的存活率服务。

急性肝衰竭起病急，预后差，临床治疗难度大。"三重打击"学说较全面地阐明了其发病机制，免疫损伤是肝细胞凋亡和坏死的首发机制，负反馈调节机制障碍是免疫调控网络紊乱的关键所在。本课题组运用流式细胞分选技术、抗原肽四聚体技术、qRT-PCR技术、免疫组化技术等实验方法，从pDCs-CTL免疫应答过程，CD4+CD25+ Treg细胞负反馈调节角度，揭示急性肝衰竭pDCs-CTL/CD4+CD25+Treg免疫网络紊乱特征及解毒化瘀颗粒的干预效应机制。同时该项目研究结果为解毒化瘀颗粒治疗肝衰竭提供技术、理论支撑,充分展示肝衰竭中医"截断逆转"法治疗理念的科学内涵,为进一步提高肝衰竭患者的存活率服务。. 项目完成明确了肝衰竭最初免疫识别过程中，外周血pDCs细胞向肝内聚集，抗原提呈与加工能力明显增强，刺激CD8+T细胞分化、增殖为效应性CTL细胞机制。成熟的DC细胞激活CTL通过穿孔素/颗粒酶系统、Fas/ FasL释放FAS、TNFR1、perforin、GrB、PD-1等凋亡因子，分泌TGF-β、IL-10、IL-4等炎症细胞因子介导肝细胞坏死与凋亡。CD4+CD25+ Treg细胞参与的负反馈调节机制障碍是急性肝衰竭免疫网络紊乱的关键因素。CD4+CD25+ Treg细胞表面膜分子CTLA-4、GITR、FoxP3、CD39通过细胞间接触方式以及释放细胞炎症因子IL-10、TGF-β、IL-4途径对特异性CD8+T的分化与增殖进行抑制，干预效应性CTL细胞功能，特异性CTL效应级联失控，诱导肝细胞大面积凋亡与坏死，打破机体的免疫平衡，最终诱导肝功能衰竭。解毒化瘀颗粒干预急性肝衰竭大鼠CD4+CD25+ Treg细胞负反馈调节机制障碍，调控机体负反馈免疫系统以恢复外周血CD4+CD25+ Treg细胞的平衡，体现了“截断”的本质，而通过不同途径防止肝细胞坏死，促进肝细胞再生，恢复肝脏组织的正常功能突显了“逆转”的寓意。解毒化瘀颗粒的干预疗效进一步明确了中医“解毒”“化瘀”的肝衰竭病机，阐明了“截断逆转法”治疗肝衰竭的科学内涵。