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  

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

作　　者：胡明冬[1] 夏世金[2] 吴俊珍 孙涛[3] 吴金峰[4] 彭正羽[5] 钱桂生[1] 

机构地区：[1]第三军医大学新桥医院呼吸内科,重庆400037 [2]复旦大学附属华东医院老年医学研究所,上海200040 [3]复旦大学附属华东医院老年医学科,上海200040 [4]复旦大学附属华山医院中西医结合科,上海200032 [5]复旦大学生物医学研究院

出　　处：《中华肺部疾病杂志（电子版）》2013年第6期4-8,共5页Chinese Journal of Lung Diseases(Electronic Edition)

基　　金：国家自然科学基金面上项目(81270115)

摘　　要：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-βGal转染再行低氧处理组(Ad-βGal+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)1 h、3 h、6 h和12 h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Western blot方法分别检测PAECs中HIF-1αmRNA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-βGal+低氧组均显著升高(P均<0.01),在低氧6 h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6 h降幅最大;②在低氧处理6 h,与常氧组比较,低氧组和Ad-βGal+低氧组HIF-1αmRNA和蛋白表达均明显上调;与低氧组或Ad-βGal+低氧组比较,Ad-Gax+低氧组HIF-1αmRNA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-1αmRNA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。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

关 键 词：GAX基因 低氧 肺动脉高压 内皮细胞 增殖 低氧诱导因子-1Α 

分 类 号：R563[医药卫生—呼吸系统]