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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 inhibitory effect of human p27kip1 gene transfection on the proliferation of vascular smooth muscle cells using nanoparticle as carrier. Method Polylactic acid polyglycolic acid copolymer was used to encapsulate the p27kip1 gene to prepare a nano-sized particle mixture. Cultured rat vascular smooth muscle cells were transfected, and the cell cycle was analyzed by flow cytometry. Animal models of autologous vein graft were established and randomly divided into transgenic group, blank vector control group and simple vein transplantation group. The thickness of intima was detected by hematoxylin and eosin (HE) and Verhoeff staining respectively. The expression of p27kip1 gene protein was detected by Western blotting. Immunohistochemistry was used to detect the expression of proliferating cell nuclear antigen (PCNA) , E2F expression. Results Compared with the other two groups, the expression of protein in the transgenic group was significantly increased at 3, 7 and 14 days (P <0.05). The mean thickness of the intima of the transgenic group was significantly lower than that of the other groups (P <0.01), and the difference between the control group and the simple vein graft group (P <0.01). The expression of PCNA in transgenic group was significantly lower than that in other groups (P <0.01), and the expression of E2F in transgenic group was significantly decreased from 7 to 14 days (P <0.01) There was no significant difference between the simple vein transplantation group (P> 0.05). Conclusion Nanoparticles can be used as transgene carrier, and the expression of p27kip1 gene can effectively inhibit the proliferation of smooth muscle cells after autologous vein grafts.