纳米粒子介导的p27^(kip1)基因转染对静脉平滑肌细胞增殖的抑制作用

The effect of p27~ (kip1)gene transfection mediated by nanoparticles on proliferation of intimal smooth muscle cells in vein graft

目的探讨以纳米粒子为载体的人p27kip1基因转染对血管平滑肌细胞增殖的抑制作用。方法聚乳酸聚乙醇酸共聚物包载p27kip1基因,制备纳米级粒子混合物。转染培养的大鼠血管平滑肌细胞,流式细胞仪分析细胞周期。建立自体静脉移植动物模型,随机分成转基因组、空白载体对照组、单纯静脉移植组。分别于术后3、7、14、28d取材,进行苏木素伊红(HE)及Verhoeff染色检测内膜厚度、Westernblot检测p27kip1基因蛋白的表达、免疫组织化学法,检测外周血增殖细胞核抗原(PCNA)、E2F表达。结果转基因组表达蛋白,3、7、14d较其他俩组明显增多(P<0.05);转基因组内膜平均厚度较其他组明显减少(P<0.01),对照组及单纯静脉移植组之间差异无统计学意义(P>0.05);转基因组PCNA的表达7～28d较其他组明显降低(P<0.01),其E2F的表达7～14d较其他组显著降低(P<0.01);对照组及单纯静脉移植组之间差异无统计学意义(P>0.05)。结论纳米粒子可以作为转基因载体,p27kip1抑癌基因的表达能够有效抑制自体静脉移植后内膜平滑肌细胞的增殖。

Objective To investigate the effect of p27~ kip1 gene transfection mediated by nanoparticles (NP) on the proliferation of intimal smooth muscle cells in rat vein grafting model.Methods Nanoparticle p27~ kip1 DNA complex was prepared with PLGA.Package efficiency,release progress invitro,and size of the complex were determined.Smooth muscle cell were transduced in vitro.The cell proliferation was determined by flow cytometry.Autogenous vein graft model was established in 120 rats by transplanting internal branch of jugular vein to carotid artery.Three groups were studied:(1)P27 group:p27~ kip1 gene mediated by NP was transfected into the veins before anastomosis;(2) Control group:The veins were transfected by simple NP;(3)Grafting group:The veins were grafted with no transfection.The grafted veins were harvested on the day 3,7,14 and 28 respectively after the operation.The exogenous p27~ kip1 protein expression in veins was detected by Western blot.Intimal hyperplasia (IH) was observed pathologically.The expression of PCNA and E2F was detected by immunohistochemistry and analyzed by computer digitizing system.Results P27~ kip1 gene transfection mediated by NP complex enabled to produce protein expression of p27~ kip1 gene (P< 0.05) and inhibit IH of the grafted vein especially for 7d-28 days significantly in p27 group (P< 0.01).Immunohistochemical analysis of PCNA indicated the decreased positive cells in the p27 group as compared with the control group at 7～28 days (P< 0.01).The expression of E2F was decreased in p27 group at 7～14 days (P< 0.01).There was no significant difference between control group and grafting group in the expression of E2F and PCNA.Conclusion NP is an effective gene transfecting carrier.p27~ kip1 gene expression can prevent IH and SMC proliferation.