低氧条件下共培养体系中肺动脉内皮细胞经Notch1/Jagged1信号通路调控肺动脉平滑肌细胞的增殖

In the hypoxic coculture system pulmonary arterial endothelial cell regulates the proliferation of pulmonary arterial smooth muscle cell via the Notch1/Jagged1 signaling pathway

目的探讨低氧条件下细胞共培养体系中肺动脉内皮细胞经由Notch1/Jagged1信号通路调控肺动脉平滑肌细胞的增殖。方法建立Transwell共培养体系,将肺动脉内皮细胞(PAEC)和平滑肌细胞(PASMC)分别接种于下室和上室,PAEC经DAPT(Notch信号阻断剂)或(和)S iR-NA-Jagged1预处理后,与PASMC共同置入自动常压缺氧孵箱进行低氧处理。根据PAEC是否行DAPT、Jagged1小RNA干扰预处理进行实验分组:常氧时PAEC与PASMC共培养对照组(N)、低氧时PAEC与PASMC共培养组(H1)、低氧时DAPT(终浓度10μmol/L)预处理PAEC与PASMC共培养组(H2)、低氧时S iRNA-Jagged1预处理PAEC与PASMC共培养组(H3)、低氧时DAPT加S iRNA-Jagged1预处理PAEC与PASMC共培养组(H4)。用3H-TdR掺入法检测PAEC和PASMC增殖情况,RT-PCR检测PAEC中Notch1、Jagged1 mRNA表达水平。结果低氧时各组PAEC和PASMC的3H-TdR掺入量明显高于常氧共培养对照组(均P<0.01);与H1组比较,H2、H3、H4组PAEC和PASMC的3H-TdR掺入量均显著降低(P<0.05,P<0.01),表明Notch1、Jagged1单阻断和双阻断使低氧条件下PAEC和PASMC增殖细胞数明显减少。低氧时PAEC与PASMC共培养组PAEC中Notch1、Jagged1 mRNA表达水平明显高于常氧共培养组(均P<0.05);用DAPT或S iRNA-Jagged1单独、或者两者共同预处理PAEC,再在低氧条件下与PASMC共培养,DAPT使PAEC中Notch1 mRNA表达水平明显下降,S iRNA-Jagged1使Jagged1 mRNA表达水平显著降低,DAPT和S iRNA-Jagged1共同使Notch1、Jagged1 mRNA表达水平同时明显下调,与H1组比较差异有统计学意义(P<0.05、P<0.01)。结论肺动脉内皮细胞表型的改变影响肺动脉平滑肌细胞表型的变化;经Notch1/Jagged1信号通路介导,低氧导致PAEC和PASMC异常增殖、低氧性肺动脉内皮细胞调控低氧性肺动脉平滑肌细胞的异常增殖。

Objective To investigate the effect of hypoxic pulmonary arterial endothelial cell （PAEC） on the proliferation of pulmonary arterial smooth muscle cell （PASMC） via the Notchl/Jaggedl signaling pathway. Methods A Transwell coculture system of PAEC and PASMC was constructed. Before hypoxic （ 3% O2 ） treatment, PASMC was put into the upper chamber while PAEC, which was pre-treated with DAPT （ inhibitor of Notch signaling） or （and） siRNA-Jaggedl, respectively, was put into the lower chamber. Transwell cocuhure systems were included the following groups： normoxie（21% O2 ）cocuhure of PAEC and PASMC （N）, hypoxic eoculture of PAEC and PASMC （H1）, hypoxic cocuhure of PAEC pretreated with DAPT and PASMC （H2）, hypoxic cocuhure of PAEC pretreated with siRNA-Jaggedl and PASMC （H3） and hypoxie coculture of PAEC pretreated with DAPT plus siRNA-Jaggedl and PASMC （H4）.^3H-TdR incorporation was used to assess the proliferation of PAEC and PASMC. The levels of Notchl and Jaggedl mRNA were estimated by RT-PCR. Results Compared with those of N group , the levels of ^3 H-TdR incorporation of PAEC and PASMC in the H1, H2, H3 and H4 groups were increased significantly （P 〈0. 01 ）. The levels of ^3H-TdR incorporation of of PAEC and PASMC in the H2 ,H3 and H4 groups were higher than those of HI group （P 〈 0. 05 ,P 〈 0. 01 ）, respectively. D-APT and siRNA-Jaggedl markedly down-regulated the expression of Notchl and Jaggedl mRNA as compared with those of H1 group（ P 〈 0. 05 ,P 〈 0. 01 ）, respectively, indicating that Notchl and Jaggedl regulate hypoxic cell proliferation. In the hypoxic coculture system PAEC influenced the proliferation of PASMC via the Notchl/Jaggedl signaling pathway. Conclusions Variation of PAEC phenotype alters PASMC phenotype. Hypoxia leads to abnormal proliferation of PAEC and PASMC, and hypoxic PAEC plays ari important role in the proliferation of hypoxic PASMC via the Notchl/Jaggedl signaling pathway.