Survivin基因过表达对血管内皮细胞增殖及抗凋亡能力的影响

Effect of 5-bromo-2-deoxyuridine labeled umbilical cord mesenchymal stem cells on cell biology behaviors in vitro

摘要:

目的：研究经转染生存素（survivin，SVV）基因后的血管内皮细胞（vascular endothelial cells，VECs）的增殖、抗凋亡能力及血管生成能力的变化。方法：携带 SVV基因腺病毒感染大鼠动脉内皮细胞（rat artery endothelial cell，RAEC）并通过 Western blot、real-time RCR的方法检测 SVV蛋白及其编码基因 birc-5。CCK8检测经 SVV干预的 RAEC其增殖能力的变化；通过过氧化氢（H2O2）诱导正常RAEC凋亡，SVV干预RAEC凋亡并通过流式细胞仪检测其凋亡率；采用Western blot法对比分析凋亡相关因子caspase 3、bcl-2及细胞周期蛋白 cyclinD含量变化；CD31标记 RAEC，通过免疫组化检测裸鼠肌肉注射 RAEC处新生血管情况。结果：SVV基因过表达后，RAEC的birc5 mRNA，SVV蛋白的表达均上调（birc5：P=0.006，SVV蛋白：P=0.000），SVV干预的凋亡 RAEC其凋亡通路中caspase 3较对照组明显降低（P=0.000），细胞周期蛋白cyclinD明显增加（P=0.000）；其增殖及血管生成能力较对照明显增高（P=0.000）。结论：SVV干预的 RAEC在抗凋亡，促增殖及血管生成方面得到了明显的提升，为血管内皮细胞（VECs）的临床应用提供一定的理论基础。

Abstract:

Objective：To observe the ability of cell proliferation，anti-apoptosis，and angiogenesis of rat artery endothelial cells （RAECs） after survivin（SVV） gene transfection. Methods：The SVV was transfected into rat artery endothelial cells（RAECs） by adenovirus. The proliferation rates of RAECs with or without SVV transfection were determined by CCK8 assay .The levels of birc5，caspase-3，bcl-2 and cyclin D before or after transfection were detected by real-time PCR and Western blot. The apoptosis of RAECs was induced by H2O2 and the apoptosis rate in each group was detected by flow cytometry. The angiogenesis was detected by immuno－histochemistry with the RAECs specific marker CD31. Results：The birc5 gene and survivin protein levels were both elevated in SVV over-expressed RAECs determined by RT-qPCR and Western-blot assay（birc5：P=0.006，SVV protein：P=0.000）. Meanwhile，the Caspase-3 protein levels were decreased and the bcl-2，cyclinD levels were increased（Caspase-3：P=0.000；bcl-2：P=0.000；cyclinD：P=0.000）. RAECs transfected with SVV exhibited a significant higher proliferation rate. The microvessel density（MVD） was much higher in RAECs transfected with SVV than in control group. Conclusion：The ability of proliferation，anti-apoptosis and angiogen－esis is significantly elevated in RAECs after SVV transfection.