基于CRISPR/Cas9技术研究抑制肝细胞necroptosis防治肝衰竭的分子机制及中医药的干预

Inhibition of Liver cell necrosis is the key and difficult point for prevention and treatment of liver failure. Previous clinical and experimental Liver failure is a complex pathogenesis, high mortality of critically ill, pathological manifestations of liver cell necrosis, inhibition of liver cell necrosis prevention and treatment of liver failure is important and difficult. Early research found SHYCT with function of prevention and treatment of liver failure, meanwhile, found hepatocytes apoptosis and necroptosis pathological features, and a significant rise in RIP3. Therefore, we surmise that liver failure happened necroptosis of RIP3 dependent. We established Acute liver failure(ALF) and Acute on chronic liver failure(ACLF) animal model by RIP3 knockout mice and normal mice,and cell injury model by CRISPR / Cas9 system overexpression and silence of RIP3 gene in normal liver cell line. In vivo and in vitro from both positive and negative angles verification RIP3 and related proteins in the liver cells necroptosis. Use immunofluorescence , western blot and qPCR to detection caspase3- RIP3 - CaMKII pathways related protein and mRNA expression. On the one hand, revealed the molecular mechanism of prevention and treatment of hepatic failure, on the other hand, to find new targets for the prevention and treatment of liver failure. At the same time ,further interpretation of that decoction multi-way,multi-target prevention and treatment of liver failure, the scientific connotation, provide sufficient theoretical basis for clinical application.

肝衰竭是发病机制复杂，死亡率高的危重病，病理表现为肝细胞坏死，抑制肝细胞坏死是防治肝衰竭的重点与难点。项目组前期研究发现三黄茵赤汤具有防治肝衰竭的作用，病理显示肝细胞具有凋亡和程序性坏死（necroptosis）的特征，且RIP3显著升高。因此，我们推测肝衰竭发生了RIP3依赖的necroptosis。拟用正常和RIP3基因敲除小鼠建立急性、慢加急性肝衰竭模型，通过CRISPR/Cas9系统技术过表达和沉默RIP3基因建立肝细胞损伤模型。体内、体外正反两个角度验证RIP3及相关蛋白在肝细胞necroptosis中的作用。免疫荧光、免疫印迹和qPCR等技术检测caspase3-RIP3-CaMKII通路相关蛋白及mRNA的表达。一方面揭示防治肝衰竭的分子机制，另一方面寻找防治肝衰竭的新靶点。同时，进一步诠释三黄茵赤汤多途径、多靶点防治急性肝衰竭的科学内涵，为临床应用提供理论依据。

目的：研究RIP3相关蛋白抑制肝细胞necroptosis防治肝衰竭的分子机制及三黄茵赤汤的干预作用。方法：正常和RIP3基因敲除小鼠通过D-氨基半乳糖联合LPS建立急性和慢加急性肝衰竭动物模型，并给予三黄茵赤汤干预；正常和RIP3基因沉默LO2肝细胞通过D-氨基半乳糖建立急性肝细胞损伤模型，并给予三黄茵赤汤粉末干预。检测小鼠肝功能和凝血功能；HE染色和电镜分析肝细胞结构和亚细胞结构的变化；免疫印迹、免疫组化和qPCR等技术分析程序性坏死通路相关蛋白及mRNA的表达。结果：与模型组比较，三黄茵赤汤高、中、低剂量减少血清ALT, AST的表达水平，缩短INR 时间，提高小鼠是生存率，改善肝脏病理损伤。三黄茵赤汤高、中、低剂量组均能减少HMGB1、TLR4、Caspase3、Caspase-1、NLRP3、RIP3、RIP1、MLKL蛋白的表达，升高Bcl-2/Bax的比值。结论：三黄茵赤汤高、中、低剂量具有防治肝衰竭的作用，并成剂量依赖。其可能机制是通过下调程序性关键蛋白RIP3并调控程序性相关蛋白，抑制肝细胞坏死，从而防治肝衰竭。