p27kip1基因表达对大鼠移植静脉内膜平滑肌细胞增殖与凋亡的影响

目的观察以聚乳酸聚乙醇酸（PLGA）纳米粒子为载体的人p27^kip1基因局部转染自体移植静脉后，对大鼠静脉内膜平滑肌细胞增殖及凋亡的影响。方法Wistar大鼠120只建立自体静脉移植模型，随机分成3组。转基因组：移植静脉转染以PLGA纳米粒子为载体的p27^kip1基因；空白对照组：转染不含有p27^kip1基因的单纯PLGA纳米粒子；单纯静脉移植组：使用生理盐水。分别于术后3、7、14、28d取材，常规HE、Verhoeff弹力纤维染色，Western blot检测p27^kip1蛋白的表达，免疫组化（SABC）法检测增殖细胞核抗原（PCNA）的表达、TUNEL法观察内膜平滑肌细胞凋亡的动态变化。结果转基因组内膜中p27^kip1蛋白表达水平高于其他组；内膜平均厚度7、14、28d低于其他2组（P〈0．01）；转基因组内膜PCNA的表达7、14d明显受到抑制（P〈0．01），平滑肌细胞的凋亡细胞百分比于7、14d较对照组明显增加（P〈0．01），单纯静脉移植组与空白对照组之间各项监测指标差异无统计学意义。结论p27^kip1基因的过表达能够有效抑制自体静脉移植后的内膜增生（IH），促进平滑肌细胞的凋亡。

The effect of p27kip1 gene transfection mediated by PLGA nanoparticles on neointimal proliferation and apoptosis in rats vein grafts

Objective To investigate whether human p27kip1 gene transfection mediated by nanoparticles( NP) can express p27kip1 protein and inhibit the neointimal formation in rat vein grafting models. Method Autogenous vein graft model was established in 120 Wistar rats by transplanting internal branch of jugular vein to carotid artery. Rats were divided into three groups: (1) p27 group; p27kip1 gene mediated by NP were transfected into the veins before anastomosis; (2) control group; the veins were transfected by simple NP; (3) grafting group;the veins were grafted without transfection. The grafted veins were harvested at 3 d, 7 d, 14 d and 28 d respectively after the operation. The exogenous p27 kip1 protein expression in veins was determined by Western blot. Intimal hyperplasia (IH) and PCNA were observed by pathology and analyzed by computer digitizing system. The presence of apoptotic VSMC was demonstrated by TUNEL method. Result p27kip1 gene transfection mediated by NP complex increased protein expression of p27kip1 gene and there was a significant decrease in the intimal average thickness at 7 d, 14 d and 28 d in p27 group (P < 0. 01). Immunohistochemical analysis of PCNA indicated decreased positive cell in the p27 group compared with the control group at 7d-28 d ( P < 0. 01). Apoptosis of VSMC in the p27 group was higher than the control group at 7 d and 14d (P <0. 01). There is no significant difference between control group and grafting group. Conclusion Over expression of p27kip1 gene prevents IH and promotes apoptosis after vein grafting.