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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 investigate the pathway of reactive oxygen species (ROS) production in rabbit vascular smooth muscle cells (VSMCs). Methods Primary cultured VSMCs were cultured with MTT method. The effects of different concentrations of hydrogen peroxide (H2O2) on the viability of VSMCs were measured. After different concentrations of 100μmol / L H2O2, the levels of superoxide anion O2-). Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the mRNA expression of NADPH oxidase subunits in VSMCs. Results As the concentration of H2O2 increased, the viability of VSMC decreased gradually. When the concentration was more than 30μmol / L, the absorbance of each cell decreased significantly (P <0.05). After treated with 100μmol / L H2O2, (1) O2 · generation in VSMC cells reached its peak at 24 h (the percentage of DHE positive cells was 24.01%); (2) the expression of p22phox mRNA increased in VSMCs, (2 times of 0 h), the expression of gp91phox and nox1 mRNA decreased. In particular, nox1 reached the lowest value at 1 h (15% of 0 h). Conclusions A certain concentration of exogenous H2O2 can promote VSMC formation. O2- and NADPH oxidase participate in this cellular effect.