2型糖尿病合并非酒精性脂肪性肝病肝细胞损伤模型的建立及探讨  

Model establishment of hepatocyte injury in type 2 diabetes mellitus complicated with nonalcoholic fatty liver disease

作　　者：勾阳阳 曾广娴 喻嵘[3] 陈聪 GOU Yangyang;ZENG Guangxian;YU Rong;CHEN Cong(Guizhou University of Chinese Medicine,Guiyang,Guizhou 550025,China;Qianxinan Autonomous Prefecture Hospital of Chinese Medicine,Xingyi,Guizhou 562400,China;Hunan University of Chinese Medicine,Changsha,Hunan 410208,China)

机构地区：[1]贵州中医药大学,贵州贵阳550025 [2]黔西南州中医院,贵州兴义562400 [3]湖南中医药大学,湖南长沙410208

出　　处：《湖南中医药大学学报》2023年第1期47-52,共6页Journal of Hunan University of Chinese Medicine

基　　金：国家自然科学基金地区科学基金项目(81860818);贵州省科学技术厅科技基金项目(黔科合基础-ZK2021:一般501);贵州省中医药管理局中医药、民族医药科学技术研究专项课题(QZYY-2022-010);2021年贵州省卫生健康委科学技术基金项目;贵州中医药大学糖脂代谢病研究中心资助项目。

摘　　要：目的探讨2型糖尿病合并非酒精性脂肪性肝病“毒损肝络”肝细胞损伤模型建立的可行性。方法将大鼠肝细胞随机分为正常组、HG组(100 mmol/L D-葡萄糖)、OA组(0.2 mmol/L OA)、HG+OA组(100 mmol/L D-葡萄糖+0.2 mmol/L OA)。干预24 h后,油红O染色观察各组肝细胞内脂滴,CCK-8法检测肝细胞的增殖情况,常规生化法检测肝细胞上清液生化指标,酶联免疫法测定肝细胞上清液NLRP-3、Caspase-1、IL-1β含量,Western blot法检测肝细胞CK8、CK18蛋白表达。结果与正常组比较,模型组脂滴蓄积明显增加;肝细胞生化指标含量均升高(P<0.05或P<0.01),肝细胞上清液中NLRP-3、Caspase-1、IL-1β含量均升高(P<0.05或P<0.01);肝细胞内CK8、CK18蛋白表达水平显著上调(P<0.05或P<0.01)。结论HG联合OA诱导肝细胞损伤建立T2DM合并NAFLD体外模型符合“毒损肝络”病机特点。Objective To explore the feasibility of establishing the hepatocyte injury model of type 2 diabetes mellitus(T2DM)complicated with nonalcoholic fatty liver disease(NAFLD).Methods Rat hepatocytes were randomly divided into normal group,HG group(100 mmol/L D-glucose),OA group(0.2 mmol/L OA)and HG+OA group(100 mmol/L D-glucose+0.2 mmol/L OA).After 24 h of intervention,lipid droplets in hepatocytes of each group were observed by oil-red O staining.The proliferation of hepatocytes was detected by CCK-8.The biochemical indexes of hepatocyte supernatant were detected by conventional biochemical method,and the levels of NLRP-3,Caspase-1 and IL-1βin it were determined by ELISA.The protein expression levels of CK8 and CK18 in hepatocytes were detected by Western blot.Results Compared with the normal group,the accumulation of lipid droplets in the model group was significantly higher.The levels of hepatocyte biochemical indexes increased(P<0.05 or P<0.01),so were those of NLRP-3,Caspase-1 and IL-1βin hepatocyte supernatant(P<0.05 or P<0.01).The protein expression levels of CK8 and CK18 in hepatocytes were significantly up-regulated(P<0.05 or P<0.01).Conclusion The establishment of an in vitro model of T2DM complicated with NAFLD induced by HG and OA is consistent with the pathogenesis of"liver-collaterals impaired by toxin".

关 键 词：2型糖尿病 非酒精性脂肪性肝病 肝细胞 CK8 CK18 NLRP3 

分 类 号：R285.5[医药卫生—中药学]