NHE-1反义RNA对大鼠肺动脉平滑肌细胞凋亡的影响

Effects of NHE-1 antisense RNA on apoptosis of pulmonary artery smooth muscle cells of rats in vitro

目的 探讨NHE 1反义RNA对大鼠肺动脉平滑肌细胞 (PASMC)凋亡的影响。方法 构建NHE 1反义RNA逆转录病毒载体 ,转染体外培养的大鼠PASMC ,应用半定量RT PCR法 (sqRT PCR)测定细胞内NHE 1mRNA的表达、BCECF法测定pHi、流式细胞仪测细胞周期、电镜及原位细胞凋亡检测 (TUNEL法 )观察细胞凋亡情况。 结果 转染NHE 1反义RNA逆转录病毒载体的细胞 ,NHE 1mRNA表达量及pHi较空载体转染细胞及未转染细胞显著降低 ,细胞凋亡率显著增加 ,电镜示细胞凋亡 ,凋亡小体形成。结论 抑制NHE 1可通过引起细胞内酸化 ,诱导肺动脉平滑肌细胞凋亡。

Objective To investigate the effects of NHE 1 antisense RNA on apoptosis of pulmonary artery smooth muscle cells(PASMC) in vitro.Methods Based on the sequence of NHE 1 mRNA,the DNA sequence encoding NHE 1 antisense RNA was obtained by PCR and subcloned into the multiple clone sites BamHI/EcoRI in retroviral vector pLXSN.Then the recombined plasmid was transfered into cultured PASMC.Expression of NHE 1 mRNA and cell cycle were detected by sqRT PCR and flow cytometric DNA analysis respectively.Intracellular pH(pHi) was measured using fluorescence dye BCECF AM.Cell apoptosis was observed with electron microscopy and terminal deoxynucleotidyl transferase medited dUTP nick end labeling (TUNEL) respectively.Results The NHE 1 mRNA expression and pHi values of cells transfected with the recombined plasmid decreased significantly than those cells transfected with pLXSN and nontransfected control.Meanwhile,the apoptosis rate of these cells increased significantly and electron microscopy also showed cell apoptosis of these cells.Conclusion NHE 1 inhibition can induce apoptosis of pulmonary artery smooth muscle cell by causing intracellular acidification.