野生型蛋白激酶GIα腺病毒抑制低氧肺动脉平滑肌细胞表型转换及细胞增殖

Adenovirus-mediated protein-kinase-GIα suppresses the hypoxia-induced proliferation and phenotype-switching of pulmonary arterial smooth muscle cell

目的 观察携带野生型蛋白激酶GIα腺病毒（Ad-PKGIα）抑制低氧诱导人肺动脉平滑肌细胞（PASMC）表型转换及其增殖的影响,探讨其在低氧肺血管重建（HPVR）中的作用.方法 组织块法建立人PASMC细胞系.Ad-PKGIα转染PASMC;采用荧光显微镜观察转染效率;RT-PCR、Western blot检测PASMC中PKGIα mRNA表达、Ad-PKGIα转染对低氧PASMC表型转换中平滑肌α肌动蛋白（SM-α-actin）表达的影响.流式细胞仪检测Ad-PKGIα转染后低氧对PASMC细胞周期的影响;~3H-TdR掺入法检测PASMC增殖.结果 （1）Ad-PKGIα转染后PASMC中PKGIα mRNA水平、磷酸化PKGIα蛋白水平显著增高.（2）常氧时PASMC中SM-α-actin蛋白表达为44.25±5.34;3% O_2处理12 h PASMC中SM-α-actin蛋白表达水平降至32.18±4.19,处理24 h SM-α-actin蛋白表达水平降至21.90±2.44.（3）低氧条件下,PASMC开始增殖,随着低氧时间延长,PASMC增殖逐渐增加,至24 h PASMC增殖最多[（14 924±1491）次/min].与未转染对照组、腺病毒空载体组比较,Ad-PKGIα转染组PASMC增殖明显受到抑制.（4）低氧使未转染对照组和腺病毒空载体组的PASMC进入有丝分裂期,随着低氧时间延长,G0/G1期细胞比例逐渐减少,S＋G_2/M期细胞比例逐渐增加.Ad-PKGIα转染后常氧状态下PASMC G0/G1期细胞比例下降、S＋G2/M期细胞比例上升趋势均明显减缓.结论 PKGIα基因在低氧肺血管重建信号转导途径中有重要的调节作用,可作为基因治疗的靶点之一.

Objective To observe the proliferation and phenotype-switching of pulmonary arterial smooth muscle cell （PASMC） induced by hypoxia and interfered by Ad-PKGIα. And to investigate the potential regulative role of PKGIα gene in the molecule mechanism of hypoxia pulmonary vessel remodeling （HPVR）. Methods To establish the pure PASMC cultured by tissue-sticking methods. Semi-quantitative reverse transcription and polymerase chain reaction （RT-PCR） and Western blot were used to examine the PKGIα mRNA and protein expression after PASMC were transfected by Ad-PKG. The mRNA and protein expressive change of smooth muscle α actin（SM-α-actin） determined the degree of cell phenotype-switching. The changes of PASMC proliferation were determined by flow cytometry and 3 H-TdR incorporated way. Results Ad-PKGIα could transfect into PASMC and highly express. Hypoxia down-regulated the expression of SM-α-actin protein （44. 25±5.34 in normoxia, 32. 18 ±4. 19 in 12 h hypoxia condition, 21.90 ±2. 44 in 24 h hypoxia condition, P 〈 0. 05 ）, that could be blocked by the transfection of Ad-PKGIct. Hypoxia could push PASMC mitosis and proliferating（3H-TdR incorporated way： 7570 ± 371 in normoxia, 12 020 ± 831 in 12 h hypoxia condition,14 924 ± 1491 in 24 h hypoxia condition, P 〈0. 05）, that could be blocked by the transfection of Ad-PKGIot, too. Conclusions The results suggested that PKGIα signaling pathway might play an important role in the molecule mechanism of HPVR. And PKGIα gene might be a target point of gene therapy.