反义雷帕霉素靶蛋白基因局部转染对移植静脉内膜增生的影响

目的:观察以纳米粒子为载体的反义雷帕霉素靶蛋白(mTOR)基因局部转染对移植静脉内膜增生的影响。方法:应用聚乳酸聚乙醇酸共聚物(PLGA)和聚乙烯醇(PVA)包载mTOR基因,制备纳米级粒子混合物。检测其包埋率、体外释放情况及粒子大小。建立自体静脉移植模型,随机分成转基因组、空载体组、对照组。转基因组移植静脉转染以纳米粒子为载体的反义mTOR基因,空载体组单纯转染纳米粒子包载的空载体,对照组不予特殊处理。分别于术后3d、7d、14d、28d取材,常规HE、Verhoeff染色,RT-PCR、Westernblot检测mTOR基因的mRNA及蛋白的变化,TUNEL法观察血管平滑肌细胞(VSMC)凋亡的动态变化。结果:转基因组内膜中mTOR基因的mRNA及蛋白产物表达较其他两组明显减少(P<0.05);转基因组内膜增生厚度7d、14d、28d较其他组明显减少(P<0.01);转基因组凋亡细胞较其他组明显增高(P<0.05)。结论:纳米粒子可以作为转基因载体,反义mTOR基因的表达能够有效抑制自体移植静脉内膜的增生,促进VSMC的凋亡。

The effect of antisense mammalian target of rapamycin gene transfection on neointimal formation in vein graft in rats

Objective:To study the effect of antisense mammalian target of rapamycin(mTOR)gene transfection mediated by nanoparticles (NP)on neointimal formation in rat vein grafts.Methods:[WTXY]Nanoparticle antisense mTOR gene complex was prepared with PLGA package.The efficiency,release process in vitro and size of the complex were determined.Autogenous vein graft model was established in 72 rats by transplanting internal branch of jugular vein to carotid artery.Three groups were studied:(1)antisense mTOR group:antisense mTOR gene mediated by NP were transfected into the veins before anastomosis.(2)Empty vector group:the vein were transfected by empty vector mediated by NP.(3)Control group(no transfection).The grafted veins were harvested 3 day,1 week,2 week and 4 week after the operation respectively.The exogenous mTOR mRNA and protein expression were determined by RT-PCR and Western blot.Intimal hyperplasia(IH) were observed by HE and Verhoeff stain.The presence of apoptotic VSMC was detected by TUNEL stain.[WTH7X]Results:[WTXY]Antisense mTOR gene transfection mediated by nanoparticle complex enabled to inhibit the mRNA and protein expression of mTOR gene(P<0.05),and the IH was inhibited in the vein graft especially during 7d~28d(P<0.01).Conclusion:Nanoparticle is an effective gene transfecting carrier,and antisense mTOR gene expression can prevent the IH and promote apoptosis after vein grafting.