摘要目的:探讨微小RNA(miRNA，miR)-33对小鼠肝再生的促进作用及其分子机制。方法:2018年9月到2019年12月，将40只C57/B6小鼠(购自上海斯莱克实验动物有限公司)按照数字随机分组法分为模型组和miR-33 KD组。模型组小鼠经尾静脉注射对照腺相关病毒1×10 11 vg/ml，miR-33 KD组小鼠经尾静脉注射miR-33敲降的腺相关病毒1×10 11 vg/ml。在注射20 d后，模型组和miR-33 KD组采用70%肝切除方法建立小鼠肝再生模型。分别在肝切术后4 d计算肝重/体重比，采用溴脱氧尿嘧啶核苷(BrdU)免疫组织化学染色分析肝脏细胞分裂能力；采用生物信息学分析miR-33的靶基因，采用蛋白质印迹法(Western blot)分析细胞增殖指标细胞核增殖抗原(Ki-67)和靶蛋白在两组肝组织中的表达。组间比较采用 t检验。 结果:模型组和miR-33 KD组小鼠术后4 d肝脏/体重比分别为(4.10±0.61)%和(4.98±0.72)%。与模型组比较，miR-33 KD组术后4 d肝脏/体重比显著增加，差异有统计学意义( t＝2.019， P<0.05)。与模型组肝脏细胞BrdU阳性率(32.47±7.99)%比较，miR-33 KD组小鼠肝脏细胞BrdU阳性率(75.38±10.56)%显著增加，差异有统计学意义( t＝4.391， P<0.05)。与模型组肝脏细胞Ki-67蛋白表达水平(0.43±0.11)比较，miR-33 KD组小鼠肝脏细胞Ki-67蛋白表达水平(1.37±0.31)显著增加，差异有统计学意义( t＝3.281， P<0.05)。生物信息学显示细胞周期素依赖性激酶(CDK6)是miR-33的靶基因。与模型组CDK6蛋白表达水平(0.25±0.15)比较，miR-33 KD组CDK6蛋白表达水平(1.02±0.28)显著增加，差异有统计学意义( t＝3.108， P<0.05)。 结论:miR-33通过靶向作用于CDK6 3’端非编码区(3’UTR)区域，调控CDK6蛋白表达水平，进而影响细胞周期，调节正常肝脏细胞增殖。

abstractsObjective:To observe the promoting effect of microRNA (miRNA, miR)-33 on liver regeneration in mice and its molecular mechanism.Methods:A total of 40 C57/B6 mice from Shanghai SLAC Laboratory Animal Co., Ltd. from September 2018 to December 2019 were randomly divided into model group and miR-33 KD group. The mice in model group were injected with 1×10 11 vg/ml of control adeno-associated virus via the caudal vein, and the mice in miR-33 KD group were injected with 1×10 11 vg/ml of adeno-associated virus knocked down by miR-33 via the caudal vein. After 20 days of injection, 70% hepatectomy was used in the model group and miR-33 KD group to establish the mouse liver regeneration model. The ratio of liver weight to body weight was calculated 4 days after hepatectomy and the ability of liver cell division was analyzed by bromo 2’-deoxyuridine (BrdU) immunohistochemical staining. The target gene of miR-33 was analyzed by bioinformatics and the expression of proliferation cell nuclear antigen (Ki-67) and target protein in the two groups was analyzed by Western blotting. T-test was used for data analysis, with P<0.05 as the difference. Results:The ratio of liver to body weight was (4.10±0.61)% in model group and (4.98±0.72)% in miR-33 KD group ( t=2.019, P<0.05). Compared with the model group, the liver/body weight ratio in miR-33 KD group significantly increased on the 4th day after operation. Compared with the model group [(32.47±7.99)%] , the positive rate of BrdU [(75.38±10.56)%] in the miR-33 KD group significantly increased ( t=4.391, P<0.05). Compared with the model group (0.43±0.11), the expression level of Ki-67 in miR-33 KD group (1.37±0.31) significantly increased ( t=3.281, P<0.05). Bioinformatics showed that Cyclin-dependent kinase 6 (CDK6) was the target gene of miR-33. Compared with the model group (0.25±0.15), the expression level of CDK6 protein in the miR-33 KD group (1.02±0.28) significantly increased ( t=3.108, P<0.05). Conclusion:MiR-33 can regulate the expression level of CDK6 protein by targeting the CDK6 3’untranslated region (3’UTR) region, thereby affecting cell cycle and regulating the proliferation of normal liver cells.