Egr-1DNA酶抑制自体移植静脉内膜增生的实验研究  

Study of Early Growth Response Gene-1 DNA Enzyme on Preventing Stenosis and Occlusion of Autogenous Vein Graft:An Experiment with Rats

作　　者：刘程伟[1] 张雪松[1] 王石[1] 田浩[1] 程明勋[1] 王树卿[1] 胡新华[2] 张强[2] 辛世杰[2] 段志泉[2] 

机构地区：[1]佳木斯大学附属第一医院普外一科,黑龙江佳木斯154003 [2]中国医科大学附属第一医院血管外科,辽宁沈阳110001

出　　处：《中国普外基础与临床杂志》2011年第10期1043-1048,共6页Chinese Journal of Bases and Clinics In General Surgery

基　　金：国家自然科学基金资助项目(项目编号:30801123;30872527)~~

摘　　要：目的探讨早期生长反应基因-1(Egr-1)DNA酶(Egr-1DNA enzyme,EDRz)对血管平滑肌细胞(VSMC)增殖和内膜增生的抑制作用,从而证实基因治疗静脉移植后狭窄、闭塞的可行性。方法构建EDRz,建立自体静脉移植模型,将大鼠右颈总静脉端-端吻合于肾下腹主动脉,EDRz转染移植静脉,分别于移植后1、2、6、24h及3、7、14、28、42d切取移植静脉标本,每个时相按随机数字表法随机抽取10只大鼠。荧光显微镜下观察EDRz转染移植静脉情况;应用原位杂交和RT-PCR方法检测Egr-1mRNA的表达;应用Western蛋白印迹和免疫组织化学方法检测Egr-1蛋白表达情况;HE染色光镜下观察组织学形态。结果①EDRz转染移植静脉情况:移植后1h时,EDRz主要位于移植静脉的外膜、中膜和部分内皮细胞;2、6及24h时,EDRz则主要位于移植静脉的中膜;7d时,EDRz主要位于移植静脉的内膜;14、28及42d时未检测到EDRz的表达。②Egr-1mRNA表达结果。RT-PCR检测结果:转染EDRz后1h时,Egr-1mRNA表达出现高峰,2、6及24h表达下降,3d时表达微弱,移植后7、14、28及42d未见Egr-1mRNA的表达,转染EDRz后1h时Egr-1mRNA表达明显高于其余各时相(P<0.01)。原位杂交检测Egr-1mRNA表达的变化趋势与RT-PCR结果基本一致。③Egr-1蛋白表达结果。Western蛋白印迹结果:正常静脉中未检测到Egr-1蛋白阳性表达。转染EDRz后2h时,出现Egr-1蛋白阳性表达,6h、24h及3d时其表达逐渐降低,移植后1h时和移植后7、14、28及42d时未见Egr-1蛋白阳性表达。移植后2h时的Egr-1蛋白表达的吸光度值高于其他时相(P<0.01)。免疫组织化学方法检测的Egr-1蛋白阳性表达的变化趋势与Western蛋白印迹结果基本一致。④EDRz转染移植静脉与未转染同期相比VSMC的增殖程度和内膜厚度明显减轻。结论 EDRz通过减少Egr-1在自体移植静脉中的表达,可有效地抑制自体移植静脉中VSMC增殖和内膜增生,可用来防治自体静脉移植后所导�Objective To detect the inhibitory effect of early growth response gene-1 DNA enzyme（EDRz） on proliferation of vascular smooth muscle cell（VSMC） and intimal hyperplasia,and confirm the effect of gene therapy on stenosis and occlusion after vein transplantation. Methods EDRz was constructed,and autogenous vein graft model was established with Wistar rats,transplanting the right jugular vein to infra renal abdominal aorta by microsurgical technique.EDRz was transfected to the graft veins and the vein graft samples were harvested at hour 1,2,6,24 and on day 3,7,14,28,42 after grafting,10 Wistar rats were randomly selected in every time.The expression of EDRz in transfected vein graft was detected by fluorescent microscope.Egr-1 mRNA was measured by reverse transcription-PCR（RT-PCR） and in situ hybridization,respectively.The protein expression of Egr-1 was detected by Western blot and immunohistochemistry,respectively.HE stained vein grafts were observed under microscope. Results ① The results of EDRz transfected vein graft： At hour 1 after grafting,EDRz was mainly located in adventitia,tunica media,and partial endothelial cells of vein graft;At hour 2,6,and 24,EDRz was located in tunica media of vein graft;and on day 7,it was mainly located in intima of vein graft.There wasn＇t EDRz in vein grafts on day 14,28,and 42.② The results of expression of Egr-1 mRNA： Detection by RT-PCR： At hour 1 after transfecting,the expression of Egr-1 mRNA arrived at the peak,and declined at hour 2,6,and 24.The expression was tenuity on day 3.Egr-1 mRNA expression was not found on day 7,14,28,and 42.The expression of Egr-1 mRNA at hour 1 was significantly higher than that of the other time point（P0.01）.The result of in situ hybridization was coincident with RT-PCR.③ The results of expression of Egr-1 protein： The result of Western blot： There was no expression of Egr-1 protein in normal veins.At hour 2 after grafting,expression of Egr-1 protein was found,and declined at hour 6,24,and on day 3.There was

关 键 词：DNA酶 移植静脉 内膜增生 

分 类 号：R654.3[医药卫生—外科学]