基于Treg/Th17细胞失衡研究清温并用法调控肝衰竭炎症反应的作用机制

The inflammation is key in the pathological mechanism of liver failure and Th17 / Treg cells is in the center of regulating liver failure inflammation ,which has presently been research highlights. It is confirmed that traditional Chinese medicine clearing and warming method(wenyangjieduhuayu decoction) is effective for the treatment of liver failure in the early ,development ,recovery stage ,which may be associated with alleviating IETM.However, the specific mechanism has been unclear. Therefore this study intends to clarify the role of Treg/ Th17 cells in regulating inflammatory response mediated by the virus and bacterial endotoxin in different stages of liver failure , by dynamically observing the frequency, transcription factors and cytokines of Treg/Th17 cell in HBV-ACLF patients peripheral blood in early ,advanced ,recovery phase. On the basic,from cellular and molecular level,the mechanism that it could regulate the imbalances of Th17 / Treg cells as for alleviating inflammatory response in liver failure will be expounded .Our team will apply to the FCM, RT-PCR, CBA experimental methods and the plasma pharmacology to intervene the Treg/Th17 cell culture in vitro experiments andendotoxin induced inflammatory response in vivo of rats ACLF model. The aim is to provide guidance for the clinical reasonable application ,the research basis and thinking for further screening and optimizing traditional Chinese medicine in the treatment.

炎症反应系肝衰竭关键病理机制， Treg/Th17细胞处于肝衰竭炎症反应调控中心，为目前研究热点。课题组前期临床研究证实中医清温并用法（温阳解毒化瘀方）对肝衰竭初期、恢复期治疗有效，基础研究提示可能与减轻肠源性内毒素血症有关，但具体机制不明。为此本研究拟通过观察HBV-ACLF患者初期、进展期、恢复期外周血Treg/Th17细胞频数、转录因子及相关细胞因子表达，阐明其在肝衰竭病毒和内毒素介导的不同炎症反应阶段的调控作用。在此基础上，通过温阳解毒化瘀方药物血浆干预Treg/Th17细胞培养的体外实验，和ACLF内毒素炎症反应大鼠模型的体内实验，采用FCM、RT-PCR、CBA技术等实验方法，从细胞、分子水平上研究清温并用法通过调控Treg/Th17细胞失衡减轻肝衰竭炎症反应的作用机制，为肝衰竭的中医药防治提供新的思路与方法。

HBV-ACLF（Hepatitis B related acute-on-chronic liver failure）属于临床危急重症，病死率极高。前期研究发现清温并用法对可减轻肝衰竭炎症反应，但对肝衰竭不同阶段炎症反应的调控机制尚不清楚。本研究首先通过临床试验，初步明确了Treg/Th17细胞免疫失衡贯穿了HBV-ACLF整个过程，失衡向Th17方向偏移，内毒素介导为主的炎症反应阶段Treg/Th17细胞表达失衡进一步加重。其次通过分离、扩增、诱导HBV-ACLF患者外周血Treg细胞和Th17细胞，建立了Treg/Th17细胞体外共培养方法，运用温阳解毒化瘀方含药血浆干预，证实了清温并用法含药血浆可降低Treg、Th17细胞频率，减少TGF-β、IL-10、TNF-α、IL-17A、IL-23细胞因子分泌，下调特异性转录因子FoxP3、ROR-γt mRNA和蛋白表达水平，促进Treg/Th17细胞向Treg方向恢复。最后采用ACLF大鼠模型，阐明了清温并用法可能通过抑制内毒素血症时Foxp3、ROR-γt的表达，从而减少外周血Treg、Th17细胞分化，促进Treg/Th17细胞恢复平衡，减轻相关促炎与抑炎因子释放对机体产生的损伤，同时抑制肝脏炎症反应，促进肝细胞损伤修复，以达到抗肝衰竭的作用。研究结果为肝衰竭的免疫调节及中医药治疗提供科学依据和新的研究方向。. 本项目共培养博士研究生1名，硕士研究生5名，发表学术论文10篇，获省部级科技成果三等奖1项。