肝脏TM6SF2特异性敲除促进非酒精性脂肪性肝病小鼠肝脏脂肪变性

Hepatocyte-specific TM6SF2 knockout aggravates hepatic steatosis in mice with nonalcoholic fatty liver disease

目的构建TM6SF2肝细胞特异性敲除的小鼠模型,研究TM6SF2在非酒精性脂肪性肝病(NAFLD)发病中的作用。方法利用CRISPR/Cas9技术和Cre/LoxP策略构建稳定的TM6SF2肝细胞特异性敲除的小鼠模型。给予TM6SF2肝细胞特异性敲除小鼠和对照小鼠普通饮食或高脂饮食喂养16周,检测小鼠体质量、肝质量等一般情况,空腹血糖、胰岛素等糖代谢相关指标,以及血浆甘油三酯、胆固醇、肝脏甘油三酯等脂代谢相关的指标。符合正态分布的计量资料两组之间比较使用t检验。结果在高脂饮食条件下,TM6SF2肝细胞特异性敲除小鼠的肝质量[(2.235±0.175)g vs(1.258±0.106)g,t=4.789,P<0.01]和肝指数(4.970%±0.298%vs 3.210%±0.094%,t=5.630,P<0.01)明显高于对照小鼠。同时,肝细胞TM6SF2基因缺失也会加重高脂饮食诱导的ALT水平异常[(62.517±1.526)U/L vs(25.991±5.947)U/L,t=5.949,P<0.01]。相较于对照组,在普通饮食和高脂饮食条件下,TM6SF2敲除小鼠血浆胰岛素水平升高[(37.203±0.836)mIU/L vs(34.835±0.426)mIU/L,t=2.520,P=0.025;(41.093±1.226)mIU/L vs(35.817±0.500)mIU/L,t=3.985,P=0.007],其他与糖代谢相关指标差异均无统计学意义(P值均>0.05)。在高脂饮食条件下,TM6SF2肝细胞特异性敲除小鼠血浆甘油三酯和胆固醇水平较对照组差异无统计学意义(P值均>0.05),而肝脏内甘油三酯水平较对照组升高[(23.969±0.978)mg/g vs(18.229±1.633)mg/g,t=3.015,P=0.024]。结论肝细胞特异性敲除TM6SF2可加重NAFLD小鼠肝脏脂质蓄积和肝损伤。

Objective To establish a mouse model of hepatocyte-specific TM6SF2 knockout,and to investigate the role of TM6SF2 in the development of nonalcoholic fatty liver disease(NAFLD).Methods The CRISPR/Cas9 technique and the Cre/LoxP strategy were used to establish a stable mouse model of hepatocyte-specific TM6SF2 knockout.The mice with hepatocyte-specific TM6SF2 knockout and the control mice were given a normal diet or a high-fat diet(HFD)for 16 weeks,and related indices were measured,including general status(body weight and liver weight),glucose metabolic indices(fasting blood glucose and insulin),and lipid metabolism(plasma triglyceride,cholesterol,and liver triglyceride).The t-test was used for comparison of normally distributed continuous data between two groups.Results Under the condition of HFD,compared with the control mice,the mice with hepatocyte-specific TM6SF2 knockout had significantly higher liver weight(2.235±0.175 g vs 1.258±0.106 g,t=4.789,P<0.01)and liver index(4.970%±0.298%vs 3.210%±0.094%,t=5.630,P<0.01),and the loss of the TM6SF2 gene in hepatocytes aggravated the abnormal level of alanine aminotransferase induced by HFD(62.517±1.526 U/L vs 25.991±5.947 U/L,t=5.949,P<0.01).Compared with the control mice under the condition of normal diet or HFD,the mice with TM6SF2 knockout had a significant increase in plasma insulin level(normal diet:37.203±0.836 mIU/L vs 34.835±0.426 mIU/L,t=2.520,P=0.025;HFD:41.093±1.226 mIU/L vs 35.817±0.500 mIU/L,t=3.985,P=0.007),while there were no significant differences in the other indices associated with glucose metabolism(all P>0.05).Under the condition of HFD,there were no significant differences in the levels of plasma triglyceride and cholesterol between the mice with hepatocyte-specific TM6SF2 knockout and the control group(P>0.05),while the mice with hepatocyte-specific TM6SF2 knockout had a significant increase in the level of liver triglyceride compared with the control mice(23.969±0.978 mg/g vs 18.229±1.633 mg/g,t=3.015,P=0.024).Conclusion Hepatocyte-specific knockout of TM6SF2 can aggravate liver lipid accumulation and liver injury in mice with NAFLD.