Egr-1在自体移植静脉中的表达及意义

Expression and Significance of Early Growth Response Gene-1 in Autogenous Vein Graft in Rats

目的研究自体静脉移植后早期生长反应基因-1(Egr-1)表达的动态变化,探讨其在内膜增生中的作用。方法Wistar大鼠90只,将大鼠右颈总静脉端-端吻合于肾下腹主动脉建立自体静脉移植模型。术后随机分别于1、2、6和24h,3、7、14、28及42d相应时间处死动物取移植静脉,同时取正常静脉作对照。应用原位杂交和RT-PCR方法检测Egr-1mRNA的表达,联合应用Western蛋白印迹和免疫组织化学方法检测Egr-1蛋白表达情况,同时进行组织形态学观察。结果自体静脉移植后,Egr-1mRNA和Egr-1蛋白的表达呈双相变化,即移植后1h,Egr-1mRNA表达迅速升高,阳性率为(35±7)%,6h、24h及3d时下降到较低水平,阳性率分别为(8±2)%、(8±6)%和(8±4)%,7d时又再升高,28d时达高峰,阳性率为(45±6)%,此与其余各时点比较差异均有统计学意义(P<0.01),42d时,Egr-1mRNA的表达再次下降;移植早期(2h)即有Egr-1蛋白的表达,阳性率为(30±5)%,并持续至6h,24h^3d表达下降到较低水平,阳性率分别为(7±3)%和(7±8)%,7d时又再升高,至移植后28d,Egr-1蛋白的表达阳性率达到高峰,为(40±9)%,此与其余各时点比较差异有统计学意义(P<0.01)。移植后7d,免疫组化结果显示,Egr-1蛋白表达主要位于中膜血管平滑肌细胞(VSMCs)和单核细胞/巨噬细胞,移植后期28d,Egr-1蛋白表达主要位于新生内膜和中膜的VSMCs,同时部分内皮细胞也有Egr-1蛋白的表达。结论移植静脉内膜增生与Egr-1的激活及表达关系密切,Egr-1可能成为防治移植静脉内膜增生、狭窄及闭塞的一个新的干预靶点。

Objective To investigate the development and significance of the expression of early growth response gene-1 （EGR-1） in autogenous vein graft in rats and detect the role of it in intimal hyperplasia. Methods Autogenous vein graft model was established in 90 Wistar rats, transplanting the right jugular vein to infra renal abdominal aorta by microsurgical technique. The vein graft samples were harvested at hour 1, 2, 6 and 24, day 3, 7, 14, 28 and 42 after procedure. Normal vein as control group. Egr-1 mRNA was measured by reverse transcription- PCR and in situ hybridization. Western blot and immunohistochemistry were used to detect the protein expression of Egr-1. Results Intimal hyperplasia reached peak at day 28 after autogenous vein graft surgery. Egr-1 mRNA and Egr-1 protein hadn＇t been found in the normal vein. The expressions of Egr-1 mRNA and Egr-1 protein had biphasic changes. By reverse transcription-PCR and in situ hybridization, we found that the level of Egr-1 mRNA rose at 1 hour after graft, the expression of Egr-1 mRNA was （35±7）%. Decline at hour 6, 24 and day 3, the positive rates of Egr-1 mRNA were （8±2）%, （8±6）% and （8±4）% respectively. Reincrease at day 7, a peak at day 28, the positive rate of Egr-1 mRNA was （45±6）% （compared with other phase, P〈0.01）. At day 42, the expression of Egr-1 mRNA declined again. Immunohistochemical staining and Western blot revealed Egr-1 protein had expressed at hour 2 early phase, the expression of Egr-1 protein was （30±5）%, and until to hour 6. The level of Egr-1 protein was decrease at hour 24 and day 3, the positive rates were （7±3）% and （7±8）% respectively. A peak at day 28, the positive rate of Egr-1 protein was （40±9）% （compared with other phase, P〈0.01）. We found that immunoreative Egr-1 located vascular smooth muscle cells （VSMCs） and monocytes/macrophages in tunica media at the early phase of day 7 and 14, and in neointimal and medial VSMCs at later phase of day 28. Egr-1 was also present in the endoluminal endothelial cells. Conclusion In autogenous vein graft, Egr-1 plays an important role in the proliferation of VSMCs. Egr-1 may become a new target for the prevention and therapy of intimal hyperplasia, stenosis and emphraxis after vein graft.