抗凋亡蛋白ARC对人主动脉血管平滑肌细胞凋亡的影响

EFFECT OF THE ANTI-APOPTOTIC PROTEIN ARC ON THE APOPTOSIS OF HUMAN AORTIC VASCULAR SMOOTH MUSCLE CELLS

目的探讨抗凋亡蛋白ARC对人主动脉血管平滑肌细胞(HA-VSMC)凋亡的影响。方法体外培养HA-VSMC,通过实时荧光定量PCR(RT-qPCR)方法检测ARC在HA-VSMC中的表达水平,以及HA-VSMC经H2O2时间梯度处理后ARC的表达量随着时间的变化;经合成的ARC过表达的腺病毒(Ad-ARC)处理HA-VSMC 24 h后,通过Western blot方法检测ARC在HA-VSMC中的过表达情况;Ad-ARC和H2O2同时处理HA-VSMC,通过caspase3/7活性检测实验和锥虫蓝染色实验检测细胞的凋亡情况。结果RT-qPCR结果显示,与H9c2、HAEC、KEK293相比,ARC在HA-VSMC中表达相对较高(F=84.50,P<0.05);RT-qPCR结果显示,在H2O2时间梯度处理以后,随着时间的推移HA-VSMC中ARC的表达量越来越低(F=8.83,P<0.05);Western blot方法检测结果显示,与对照组和β-gal组进行比较,Ad-ARC组HA-VSMC中ARC的表达量显著上调(F=296.00,P<0.01);caspase3/7活性检测实验和锥虫蓝染色实验检测结果显示,与经过H2O2处理组相比,Ad-ARC明显抑制了H2O2诱导的HA-VSMC的凋亡(F=55.46、190.10,P<0.05)。结论抗凋亡蛋白ARC在HA-VSMC中高表达,过表达ARC可抑制H2O2诱导的HA-VSMC的凋亡。

Objective To investigate the effect of the anti-apoptotic protein ARC on the apoptosis of human aortic vascular smooth muscle cells(HA-VSMCs).Methods HA-VSMCs were cultured in vitro,and quantitative real-time PCR(RT-qPCR)was used to measure the expression of ARC in HA-VSMCs and the change in the expression of ARC after treatment with H2O2(time gradient).HA-VSMCs were treated with the synthesized adenovirus with ARC overexpression(Ad-ARC)for 24 h,and then Western blot was used to investigate the overexpression of ARC in HA-VSMCs.HA-VSMCs were treated with Ad-ARC and H2O2,and then caspase 3/7 activity assay and Trypan blue staining were used to observe cell apoptosis.Results RT-qPCR results showed that compared with H9c2,HAEC,and KEK293 cells,HA-VSMCs had significantly higher expression of ARC(F=84.50,P<0.05).After the treatment with H2O2(time gradient),the expression of ARC in HA-VSMCs gradually decreased over the time of treatment(F=8.83,P<0.05).Western blot showed that compared with the control group and theβ-gal group,the Ad-ARC group had a significant increase in the expression of ARC in HA-VSMCs(F=296.00,P<0.01).The results of caspase 3/7 activity assay and Trypan blue staining showed that compared with the H2O2 treatment group,Ad-ARC significantly inhibited the apoptosis of HA-VSMCs induced by H2O2(F=55.46,190.10,P<0.05).Conclusion The anti-apoptotic protein ARC is highly expressed in HA-VSMCs,and overexpression of ARC can inhibit the apoptosis of HA-VSMCs induced by H2O2.