肾脏过表达GJB1基因动物模型的构建

Establishment of mouse models of renal overexpression of GJB1 gene

目的构建小鼠过表达GJB1基因的重组腺相关病毒(rAAV)载体,建立一种小鼠肾脏过表达GJB1基因的动物模型。方法采用PCR扩增制备小鼠GJB1基因片段,利用基因重组技术构建含GJB1基因的重组质粒,将携带小鼠GJB1基因的腺相关病毒(AAV)穿梭质粒与pAAV-RC、pHelper共转染AAV-293细胞,通过选择培养基筛选出同源重组的阳性克隆获得rAAV。将30只成年C57BL/6J雄性小鼠随机分为5组各6只(W1组、W2组、W3组、W4组及空白对照组),经尾静脉将rAAV注射到W1~W4组小鼠体内,分别于注射后1周、2周、3周、4周取其心脏、肝脏、肾脏组织行PCR检测GJB1 mRNA的表达情况,并经石蜡包埋切片后采用免疫组织化学法检测Cx32的表达。结果含GJB1基因的rAAV被成功构建。随着注射后时间的延长,小鼠肾脏组织GJB1基因转录及Cx32蛋白表达强度均逐渐增强,与空白对照组比较差异有统计学意义(P均<0.05),而其他器官(肝脏、心脏)GJB1基因表达未见明显增强(P均>0.05)。结论采用rAAV技术可成功构建肾脏过表达GJB1基因的小鼠模型,这对深入研究GJB1基因在肾损伤中的作用机制、评估以GJB1为治疗靶点的药物疗效等具有重要的科学价值和临床意义。

Objective To construct a recombinant adeno-associated virus vector(rAAV)overexpressing GJB1 gene,and to establish a mouse model overexpressing GJB1 gene in the kidney.Methods GJB1 gene fragment was amplified by PCR.Recombinant plasmids containing GJB1 gene were constructed by gene recombination technology.AAV carrying GJB1 gene was co-transfected with pAAv-RC and pHelper into AAV-293 cells.Homologous recombinant postive clones were screened to obtain rAAV.Thirty adult male C57BL/6J mice were randomly divided into five groups(W1,W2,W3,W4 and blank control groups),six in each group.rAAV was injected into the mice in the W1-W4 groups through the tail vein.The heart,liver and kidney tissues were taken at 1-,2-,3-and 4-week after injection,respectively.The expression level of GJB1 mRNA ws quantitatively measured by PCR.The expression level of Cx32 mRNA was detected by immunohistochemistry after paraffin embedded sections.Results The rAAV containing GJB1 gene was successfully constructed.With the prolonged time after injection,the GJB1 gene transcription and expression intensity of GJB1 protein in renal tissues were gradually increased,which significantly differred from those in the blank control group(both P<0.05).However,the expression intensity of GJB1 gene in other organs,such as liver and heart,did not increase significantly(all P>0.05).Conclusions The mouse model with kidney-specific over-expression of GJB1 gene can be successfully constructed by the rAAV technology,which has important scientific value and clinical significance for in-depth study of the mechanism of GJB1 gene in renal injury and for the evaluation of drug efficacy with GJB1 as the therapeutic target.