病毒载体对大鼠移植静脉的影响

Effects of adenovirus on vein autografts:experiment with rat

目的探讨腺病毒载体转染后对大鼠移植静脉的影响。方法选用 Wistar 大鼠间置移植颈静脉于同侧颈总动脉,术后移植静脉行 HE 染色和 VG 染色,观测血管内膜厚度。应用免疫组织化学染色,观察血管内膜平滑肌细胞(SMC)内增殖细胞核抗原(PCNA)表达及增殖情况。于倒置荧光显微镜,以紫外光激发观察测定腺病毒转染率。逆转录聚合酶链反应(RT-PCR)及 Western 蛋白印迹检测细胞间黏附分子-1(ICAM-1)、血管细胞黏附分子-1(VCAM-1)水平。结果术后3 d,GFP表达较高;术后10 d,GFP 表达有所下降;术后30 d,GFP 几乎没有表达。移植术后10 d 试验组及对照组可见明显内膜增生。移植术后30 d 试验组及对照组内膜增生减轻。试验组及对照组与正常非移植静脉相比,内膜增生明显(P<0.05),试验组较对照组内膜增生稍明显,但差异无统计学意义。实验中正常静脉平滑肌细胞 PCNA 阳性细胞极少,对照组与试验组术后10 d,内膜与中膜 SMC 的PCNA 阳性细胞增多,增殖达到高峰。术后30 d 中膜 SMC 的 PCNA 阳性细胞明显减少。试验组及对照组与正常非移植静脉相比,PCNA 阳性细胞增高明显(P<0.05)。试验组较对照组 PCNA 阳性细胞稍多,但差异无统计学意义。正常静脉 ICAM-1、VCAM-1的 mRNA 和蛋白表达很少,对照组移植后3 d 部分细胞即出现阳性表达,10 d 表达最强,30 d 仍较高。与正常静脉组比较,差异有统计学意义(P<0.05)。试验组较对照组表达更高,但与对照组比较,差异无统计学意义。结论腺病毒载体能高效地进行体内基因转移,作用时间短暂。腺病毒载体促进移植静脉的血管内 SMC 活化、增多、血管内膜增生、ICAM-1、VCAM-1的表达的增加,腺病毒转染到移植静脉血管壁后产生炎症、内膜增生等效果,混淆了目的基因的效果。

Objective To assess the effects of transfection of adenovirus on the tunica intima hyperplasia of vein autografts. Methods An external branch of the internal jugular vein, 5 mm in length, of a Wistar rat was cut and transplanted to its own common carotid artery by end to end bypass so as to establish a model. Ninety Wistar rats underwent transplantation of vein autografts to their own arteries and then were randomly divided into 5 equal groups： experiment group, undergoing transplantation and adenovirus transfection; experimental control group, undergoing transplantation only; and normal control group. Three, 10, and 50 days later, samples of vessel were obtained. The expression of green fluorescence protein （GFP） was observed so as to measure the transfection rate of adenovirus. Routine HE staining and Verheoff Van Gieson staining were made to measure the thickness of the vessels with computer-assisted image analyzer. Immunohistochemistry was used to detect the proliferating cell nuclear antigen （PCNA） RT-PCR and Western blotting were used to detect the mRNA and protein expression of intracellular adhesion molecule-1 （ ICAM-1 ） and vascular adhesion molecule-1 （ VCAM-1 ）. Results Three day postoperatively, the expression of GFP reached the peak, （ 26. 4 ± 3.6 ） % ; decreased to （ 14. 5 ± 2. 1 ） % 10 day postoperatively; and became very low 30 day postoperatively, at the level of （0. 81 ± 0. 2 ）%. The hyperplasia of venous tunica intima of the control group was obvious 10 day postoperatively, and became less obvious 30 day postoperatively. Proliferation of vascular smooth muscle cells （VSMCs） and sedimentation of extra-cellular matrix in the experimental group were very obvious , compared with the other 2 groups （ both P 〈0.05）. PCNA was very rare in normal vein walls, showing that the VSMCs were in the static phase. PCNA presentation increased obviously in the control group, showing a high proliferation rate of VSMCs. The positive rate of PCNA was associated with the thickness of the tunica intima, mRNA expression and protein expression of ICAM-1 and VCAM-1 could be detected 3 days after the transplantation, peaked 10 days after the transplantation, and remained high 30 days after in the normal group, however, the corresponding expression levels of the normal vein group were all very low （ all P 〈 0.05 ） . The corresponding levels of the experimental group were all higher than those of the experimental control group, however, not significantly different. Conclusion Transfection of adenovirus into the wall of transplanted vein causes inflammation and hyperplasia of tunica intima. However, such affects only a short time.