摘要
目的探讨p120-连环蛋白(p120cnt)表达对急性肝衰竭(ALF)内皮损伤和炎性激活的影响,分析p120cnt的抗炎效应是否可以通过对Epac1信号通路的调控来发挥作用。方法查询GenBank中大鼠p120cnt cDNA文库,设计引物,PCR扩增目的基因,琼脂糖电泳鉴定结果。经酶切、连接克隆至带绿色荧光蛋白(GFP)的表达载体,转化E.Coli JM109并筛选阳性菌落,提取重组质粒后酶切,测序验证,确保表达框无误。选择健康雄性Wistar大鼠36只,按随机数字表法分为正常对照组、ALF模型组、质粒转染+ALF建模组,每组12只。脂多糖(LPS)联合D-GalN法构建ALF动物模型,使用裸质粒流体力学基因转染法转染大鼠,肝组织切片荧光显微镜观察转染是否成功。以上处理12h后处死大鼠取肝组织切片进行HE染色及TUNEL分析,评估各组肝细胞坏死程度;半定量RT-PCR评估各组肝组织p120cnt mRNA及Epac1mRNA表达水平。结果 PCR扩增目的基因,1.2%琼脂糖电泳显示目的基因约2800bp。真核表达重组体pCMV/p120ctn提取质粒后酶切,酶切产物基因测序显示质粒构建正确。基因转染后大鼠肝组织切片内可见大量GFP表达说明转染已成功。大鼠肝组织切片HE染色及TUNEL分析说明质粒转染+ALF建模组肝细胞坏死严重程度低于ALF模型组。大鼠肝组织p120cnt mRNA表达强弱顺序为:正常对照组>质粒转染+ALF建模组>ALF模型组(均P<0.01)。大鼠肝组织Epac1mRNA表达强弱顺序为:质粒转染+ALF建模组>ALF模型组>正常对照组(P<0.05或P<0.01)。结论 p120cnt存在对ALF的抗炎作用和肝保护作用;p120cnt对肝组织急性炎症的抑制效应与上调Epac1基因表达有关。
Objective To investigate the effects of p120-catenin(p120cnt)expression on endothelial injury and inflammatory activation in acute liver failure(ALF),and to analyze whether the anti-inflammatory effect of p120cnt is mediated by Epac1 signaling pathway.Methods Rat p120cnt cDNA was isolated from GenBank library.PCR primers were designed to amplify the target gene,and results were identified by agarose gel electrophoresis.After enzyme digestion,the fragment was cloned into GFP-expressing vector.The recombinant plasmids were transfected into E.coli JM109 and positive colonies were screened and sequenced to ensure correct expression.Thirty-six male Wistar rats were randomly divided into normal control group,ALF model group and plasmid transfection+ALF model group,with 12 rats in each group.Lipopolysaccharide combined with D-GalN was used to construct ALF animal model.The result of hydrodynamics-based transfection was evaluated by observing liver tissue sections under fluorescence microscopy.After 12 hours,rats were sacrificed and liver tissue sections were stained with HE and TUNEL to assess the degree of hepatocellular necrosis.The expression of p120cnt and Epac1 mRNA in liver tissue was measured by RT-PCR.Results The 1.2%agarose electrophoresis showed that the target gene was about 2800 bp.Gene sequencing of enzyme-digested products of eukaryotic expression recombinant pCMV/p120ctn showed that the plasmid was constructed correctly.A large amount of GFP expression was found in the liver tissue sections suggested that the transfection was successful.HE staining and TUNEL analysis showed that the severity of hepatocellular necrosis in plasmid transfection+ALF model group was lower than that in ALF model group.Compared with plasmid transfection+ALF model group,the expression of p120cnt mRNA increased in normal control group but decreased in ALF model group(P<0.01).Compared with ALF model group,the expression of Epac1 mRNA increased in plasmid transfection+ALF model group but decreased in normal control group(P<0.05 or P
作者
余子琪
陈蕾
章慧芳
陈建勇
YU Zi-qi;CHEN Lei;ZHANG Hui-fang;CHEN Jian-yong(Department of Gastroenterology,Jiangxi Provincial People’s Hospital, Nanchang 330006,China)
出处
《南昌大学学报（医学版）》
CAS
2017年第6期6-11,共6页
Journal of Nanchang University：Medical Sciences
基金
江西省科技厅重点研发计划一般项目(20171BBG70087)
江西省卫生计生委科技计划(20171002)
