shRNA干扰Keap1对缺氧肝细胞功能的影响  

Effects of Keap1 shRNA interference on the functions of hypoxic hepatocytes

作　　者：刘静[1] LIU Jing(Department of Pathology,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China)

机构地区：[1]郑州大学第一附属医院病理科,郑州450052

出　　处：《临床与实验病理学杂志》2022年第9期1025-1029,共5页Chinese Journal of Clinical and Experimental Pathology

基　　金：河南省医学科技攻关计划联合共建项目(2018020081)。

摘　　要：目的探讨短发夹RNA(small hairpin RNA,shRNA)干扰肝细胞内氧化应激感受器Keap1下调后对缺氧肝细胞功能的影响。方法构建4条Keap1 shRNA(Plvx/GFP/puro-Keap1-1781、1894、1934、2097)和1条阴性对照shNC质粒序列,并筛选干扰效率最高的序列转染至小鼠肝细胞AML12中,采用Western blot技术检测不同缺氧处理时间Keap1以及其效应分子Nrf2蛋白水平,并通过测定Ⅲ型胶原α1(COL3A1)、TGF-β1、VEGF-A和IGF-1的表达了解其对促纤维化分子的影响。应用ELISA法测定上清中COL3A1的含量;运用WST-1检测缺氧肝细胞的活性;采用丙酮酸激酶(PK)活性及ATP含量测定判断缺氧肝细胞能量代谢状态。结果转染Plvx/GFP/puro-Keap1-1781、1894、1934、2097后,Keap1 mRNA相对表达量分别为0.38±0.16、0.42±0.14、0.62±0.17、0.50±0.13,Plvx/GFP/puro-Keap1-1781质粒序列干扰效率最高,应用其转染至缺氧肝细胞AML12后,Keap1蛋白水平明显下降(P<0.05),Nrf2表达上升(P<0.05)。与对照组相比,COL3A1、TGF-β1、VEGF-A和IGF-1蛋白表达受到抑制,且ELISA检测显示COL3A1分泌量减少(P=0.002)。Keap1 shRNA干扰后,与对照组相比,缺氧肝细胞的活性上升(12 h:P=0.048;24 h:P=0.045;48 h:P=0.032),PK活性(P=0.045)和ATP含量升高(P=0.041)。结论转染Keap1 shRNA可降低缺氧肝细胞中促纤维化分子的表达,减少缺氧对肝细胞的损伤,为临床防治肝纤维化提供新的靶点。Purpose This study is designed to investigate the effect of intracellular oxidative stress sensor Kelch-like ECH-associated protein 1(Keap1)on hypoxic hepatocytes using small hairpin RNA(shRNA)method.Methods Four Keap1 shRNAs(Plvx/GFP/puro-Keap1-1781,1894,1934,2097)and one negative control(shNC)plasmid sequence were constructed and the sequence with the highest interference efficiency was screened for transfection into moue hepatocytes AML12.The level of Keap1 and its effector molecule Nrf2 protein were analyzed by Western blot at different hypoxia treatment times.And the expression of collagen typeⅢα1(COL3A1),TGF-β1,VEGF-A,and IGF-1 were detected to understand their effects on pro-fibrotic molecules.The ELISA was used to determine the level of COL3A1 in the supernatant of transfected cells.WST-1 assay was used to measure the hypoxia hepatocytic cells viability,the energy metabolism of hypoxic hepatocytes was assessed by determination of Pyruvate kinase(PK)activity and ATP content.Results Plvx/GFP/puro-Keap1-1781,1894,1934,2097 were transfected into AML12 cells,the expression of Keap1 mRNA were 0.38±0.16,0.42±0.14,0.62±0.17,0.50±0.13.The plasmid sequence interference of Plvx/GFP/puro-Keap1-1781 was the most efficient,and application of ite transfection to hypoxic hepatocytes AML12 resulted in a significant decrease in Keap 1 protein levels and an increase in Nrf2 expression(P<0.05).The protein expression levels of COL3A1,TGF-β1,VEGF-A,and IGF-1 in AML12 cells were significantly decreased in comparison to that of control group.ELISA showed the decreased COL3A1 expression(P=0.002)in the Keap1 shRNA-transfected cells.Furthermore,the transfected cells showed higher cellular activity(12 h:P=0.048,24 h:P=0.045,48 h:P=0.032),enhanced PK activity(P=0.045)and high ATP content(P=0.041).Conclusion Keap1 shRNA transfection may decrease the expression of pro-fibrotic molecules proteins,and ameliorating hypoxic injury in hepatocytes,which offers a new therapeutic strategy for liver fibrosis.

关 键 词：肝纤维化 肝细胞 KEAP1 NRF2 

分 类 号：R333.4[医药卫生—人体生理学]