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目的应用RNA干扰抑制肺动脉平滑肌细胞(pulmonary aretery smooth muscle cells,PASMCs)基质交感分子1(stromal interaction molecule 1,STIM1)基因表达,探讨STIM1表达抑制后对PASMCs缺氧增殖反应的影响。方法采用离体培养大鼠肺动脉平滑肌细胞,设对照组、阴性siRNA组(SiNT)及阳性siRNA组(SiSTIM1),低氧(3%O2)培养24 h,Western blot法检测PASMCs中STIM1基因表达变化,MTT比色法及[3H]-TdR掺入法检测PASMCs的增殖情况,流式细胞仪检测PASMCs的细胞周期变化。结果 Western blot检测SiSTIM1组STIM1蛋白相对灰度值为(0.355±0.008),显著低于对照组(0.624±0.017)和SiNT组(0.618±0.021)(P<0.05)。MTT比色实验及[3H]-TdR掺入实验显示低氧培养24 h后,对照组和SiNT组PASMCs较常氧组增殖显著(P<0.05),而SiSTIM1组PASMCs增殖活性较对照组和SiNT组显著降低(P<0.05)。流式细胞仪检测结果表明低氧培养24 h后,对照组和SiNT组PASMCs G0/G1期细胞比例较常氧组显著降低(P<0.05),而SiSTIM1组G0/G1期细胞比例则显著高于对照组和SiNT组(P<0.05)。结论 RNA干扰介导STIM1基因沉默可明显抑制低氧下大鼠肺动脉平滑肌细胞增殖。
Objective To investigate the effect of STIM1 expression on the hypoxia-induced proliferation of PASMCs in vitro by RNA interference (RNAi) inhibition of stromal interaction molecule 1 (STIM1) gene expression in pulmonary atery smooth muscle cells (PASMCs). Methods Rat pulmonary artery smooth muscle cells (VSMCs) were isolated and cultured in vitro. SiRNAs, SiRNAs and SiRNAs were transfected into the smooth muscle cells of rats for 24 h. The expression of STIM1 in PASMCs was detected by Western blot. MTT assay and [3H] -TdR incorporation were used to detect the proliferation of PASMCs. The cell cycle changes of PASMCs were detected by flow cytometry. Results Compared with control group (0.624 ± 0.017) and SiNT group (0.618 ± 0.021), the relative gray value of STIM1 protein in SiSTIM1 group was (0.355 ± 0.008) weeks by Western blot (P <0.05). MTT colorimetric assay and [3H] -TdR incorporation assay showed that PASMCs in control group and SiNT group proliferated more significantly than that in normoxia group (P <0.05) 24 h after hypoxia, while the proliferative activity of PASMCs in SiSTIM1 group was significantly higher than that of control group and SiNT Group was significantly lower (P <0.05). The results of flow cytometry showed that the proportion of cells in G0 / G1 phase of PASMCs in control group and SiNT group was significantly lower than that in normoxia group (P <0.05) after 24 h hypoxia incubation, while the proportion of cells in G0 / G1 phase in SiSTIM1 group was significantly higher In control group and SiNT group (P <0.05). Conclusion Silencing of STIM1 by RNA interference can significantly inhibit the proliferation of pulmonary artery smooth muscle cells in hypoxia rats.