兔血管平滑肌细胞活性氧的生成途径

Pathway of reactive oxygen species production in rabbit vascular smooth muscle cells

目的探讨兔血管平滑肌细胞(VSMC)生成活性氧的途径。方法兔VSMC原代培养,噻唑蓝(MTT)法检测不同浓度过氧化氢(H2O2)对VSMC活力的影响;100μmol／L H2O2作用后不同时段,流式细胞仪检测VSMC细胞内超氧阴离子(·O2-)生成;逆转录-聚合酶链反应(RT- PCR)检测VSMC NADPH氧化酶各亚基mRNA表达。结果随H2O2浓度增加,VSMC细胞活力逐渐下降,浓度≥30μmol／L时,各时段吸光度值即有显著降低(P<0．05)。100μmol／L H2O2作用后,(1)VSMC细胞内·O2-生成,其作用在24 h达峰值(DHE阳性细胞百分率24．01％);(2)VSMC p22phox mRNA的表达增加,并在1 h达峰值(为0 h的2倍),gp91phox和nox1 mRNA的表达下降,尤其nox1,在1 h达最低值(为0 h的15％)。结论一定浓度的外源性H2O2可促进VSMC生成·O2-,NADPH氧化酶参与这一细胞效应。

Objective To study the pathway of reactive oxygen species （ROS） production in rabbit vascular smooth muscle ceils （VSMCs）. Methods Primary cell culture of VSMCs and MTT assay were performed to detect the cell viability after being treated with different concentrations of H2O2. 100 μmol/L H2O2 was used on VSMC as the final concentration, intracellular · O2^- production was measured by flow cytometry and mRNA expression of subunits of VSMC NADPH oxidase was detected by RTPCR at different time points after treatment. Results VSMC viability was decreased gradually with the increase of the concentration of H2O2. When the concentration 930 μmol/L, the A value at each time point was decreased significantly （P〈 0.05）. Under the condition of 100 μmol/L H2O2, intracellular · O2^- production was increased and reached the peak value at 24th h after treatment （24.01% of DHE- positive cell rates） ;the expression of p22phox mRNA was increased gradually and reached the peak value 1 h after treatment （2 times of 0 h）, while gp91phox and noxl mRNA expression decreased, especially the expression of noxl mRNA decreased down to the lowest 1 h after treament （as 15 % as 0 h）. Conclusion Certain concentration of exogenous H2O2 can contribute to VSMC · O2 ^- prduction, and NADPH oxidase may involves in these cellular events.