Gax基因双向调节低氧性肺动脉内皮细胞增殖及影响HIF-1α基因表达的研究

目的观察Gax基因对低氧性肺动脉内皮细胞（PAECs）增殖和低氧诱导因子-1α（HIF-1α）基因表达的影响，为进一步研究Gax基因调节低氧性肺动脉高压（HPH）的作用与机制奠定基础。方法取大鼠肺动脉，用酶消化法获取PAECs并进行原代培养；PAECs分4组：未转染常氧对照组（常氧组）、未转染低氧处理组（低氧组）、Ad—SGal转染再行低氧处理组（Ad—SGal＋低氧组）、Ad—Gax转染再行低氧处理组（Ad—Gax＋低氧组）。分别在常氧（21％O2）和低氧（2．5％O2）1h、3h、6h和12h各时相点，采用3H-胸腺嘧啶核苷（3H-TdR）掺入法检测PAECs增殖；使用RT—PCR和Weatern blot方法分别检测PAECs中HIF-1α mRNA和蛋白表达水平。结果①PAECs的3H-TdR掺入量：与常氧组同时相点比较，低氧组和Ad—pGaJ＋低氧组均显著升高（P均〈0．01），在低氧6h达最大值；Ad．Gax＋低氧组与常氧组同时相点比较均显著升高（P均〈0．01），但与低氧组比较却均明显降低（P〈0．01、P〈0．05），到低氧6h降幅最大；②在低氧处理6h，与常氧组比较，低氧组和Ad—BGal＋低氧组HIF-1α mRNA和蛋白表达均明显上调；与低氧组或Ad—BGal＋低氧组比较，Ad—Gax＋低氧组HIF-1α mRNA和蛋白表达皆显著下调，差异有统计学意义（P〈0．05、P〈0．01）。低氧早期内皮细胞异常增殖加速，而此时增强Gax基因的表达可抑制细胞的异常增殖；随着低氧时间的不断延长，细胞增殖受到抑制，而此时增强内皮细胞中Gax基因表达却又促进细胞增殖，以此来维持细胞的数量。结论Gax基因对维持内皮细胞数量的稳态具有双向调节作用。增强Gax基因的表达能下调低氧诱导的HIF-1α mRNA和蛋白表达，这可能与Gax基因抑制低氧性内皮细胞异常增殖的机制相关。

Effects of Gax gene on proliferation and HIF-1a gene expression of hypoxic pulmonary arterial endothelial cells

Objective To study the effect of Gax gene on proliferation of hypoxic pulmonary arterial endothelial cells and HIF-lctgene expression, in order to investigate the role of Gax gene in anti-hypoxic pulmonary arterial hypertension. Methods Pulmonary arterial endothelial cells （PAECs） were obtained from rat pulmonary artery by enzymatic digestion and were primarily cultured. PAECs were divided into 4 groups： non-transfected normoxia （normoxia group）, untransfected hypoxic treatment group （hypoxia group）, Ad-βGal transfected and hypoxic treatment group （Ad-βGal ＋ hypoxia group ）, Ad-Gax transfection and hypoxic treatment group （Ad-Gax ＋ hypoxia group） . Under conditions of normoxia （21% 02 ） and hypoxia （2.5% O5） for 1 hour, 3 hours, 6 hours and 12 hours, cells proliferation was assessed by 3H-thymidine （3H-TdR） incorporation assay. The levels of HIF-1a mRNA and protein were evaluated with RT-PCR and Western blot at the 6-hour-hypoxia treatment, respectively. Results ①3H-TdR incorporation of PAECs： Compared with the normoxia group, the 3H-TdR incorporation of PAECs was significantly increased in the hypoxia group and Ad-βGal ＋ hypoxia group in various time points （ P 〈 0.01 ）. And it reached the maximum at 6-hour-hypoxia time point. The 3H-TdR incorporation of PAECs in the Ad-Gax ＋ hypoxia group was higher than those in the normoxia group （ P 〈 0.01 ） and lowed than the hypoxia group （ P 〈 0.01, P 〈 0.05 ）. The largest decline occurred at 6-hour-hypoxia time point; ②Expression of HIF-1 ctmRNA and protein： At the 6-hour-hypoxia treatment, compared with the normoxia group, the levels of HIF-1a mRNA and protein were up-regulated in the hypoxia group and Ad-βGal ＋ hypoxia group. Whereas the levels of HIF-lotmRNA and protein were down- regulated in the Ad-Gax ＋ hypoxia group as compared to the hypoxia group and Ad-βGal ＋ hypoxia group （P 〈 0.05, P 〈 0.01 ）. Hypoxia in the early stage accelerates endothelial cell proliferation, but this time forced Gax gene expression can inhibit abnormal cell proliferation. With the increasing of hypoxie time, cell proliferation is inhibited, hut this time forced Gax gene expression in endothelial cells activates cell proliferation, thereby to maintain the cell number. Conclusions Gax plays a dual regulatory role in maintaining endothelial cells in number homeostasis. Force Gax gene expression can down-regulate hypoxia-indueed expression of HIF- 1 ctmRNA and protein, which may relate to role of Gax gene in inhibiting hypoxia-induced PAECs proliferation.