腺病毒载体介导肝细胞生长因子基因对冠状动脉再狭窄的预防作用

Effect of Adenovirus Vector Mediated Hepatocyte Growth Factor Gene Transfection on Coronary Artery Restenosis

目的应用重组腺病毒载体介导的肝细胞生长因子(HGF)基因转染血管内皮细胞(VEC)及血管平滑肌细胞(VSMC),探讨HGF基因对其凋亡的诱导作用。方法以肝细胞中抽提的总RNA为模板,反转录cDNA,采用RT-PCR技术获得HGF基因,并引入酶切位点,转染大肠埃希菌,筛选阳性菌落,经酶切及测序证实后,转染腺病毒,制造病毒包涵体,经3轮扩増,制备高效表达HGF基因腺病毒载体(Ad-HGF)。以此感染VEC及VSMC,设为Ad-HGF组;再以人工合成HGF培养VEC及VSMC为阳性对照组(HGF组);以未感染Ad-HGF的细胞为阴性对照(对照组)。并采用流式细胞仪检测缺氧情况下各组细胞的凋亡率。结果VEC的Ad-HGF、HGF组细胞凋亡率均低于对照组,差异均有极显著性意义(均P<0.01),但VSMC的3组细胞凋亡率差异无显著性意义(P>0.05)。结论腺病毒载体介导HGF基因感染VEC后能在缺氧情况下有效地阻止VEC发生凋亡,但不能有效抑制VSMC凋亡,提示对冠状动脉再狭窄有预防作用。

Objective To investigate transfection of hepatocyte growth factor （Ad-HGF） gene into vascular endothelial cells （VECs） and vascular smooth muscle cells （VSMCs） mediated by adenovirus vector and explore the induction of apoptosis by HGF gene. Methods Total RNA was extracted from human hepatocytes as primer and reversely transcribed into cDNA. By using RT-PCR HGF gene was obtained and transfected into E. coll. The positive clones were slected, identified by enzyme digestion and sequencing, and transfected into adenovirus to produce viral inclusion bodies. After amplification by 3 circles, highly expressed Ad-HGF vectors were made. Ad-HGF was transfected into VECs and VSMCs as Ad-HGF group; VECs and VSMCs were cultured with HGF as positive control group （HGF group）; and the cells not infected with Ad-HGF as negative controls （control group）. The rate of apoptosis was detected by flow cytometry in all groups under anoxic conditions. Results In Ad-HGF group and HGF group, the apoptosis rate of VECs was significantly lower than in control group （both P〈0.01）. However, there was no significnt difference in the apoptosis rate of VSMCs among three groups （P〉0. 05）. Conclusion Transfection of Ad-HGF into VECs can effectively block the apoptosis of VECs under anoxic conditions, but can＇t effectively suppress the apoptosis of VSMCs, suggesting transfection of Ad-HGF can prevent coronary artery restenosis.