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目的区分大鼠缺氧性肺动脉高压时肺血管壁3种缺氧诱导因子α(HIFα)亚基(HIF1α、HIF2α、HIF3α)的基因表达特征。方法40只雄性Wistar大鼠按随机数字表法分为常氧0d组(H0组)、缺氧3d组(H3组)、7d组(H7组)、14d组(H14组)和21d组(H21组),每组8只。用(10.0±0.5)%的氧浓度每天间断缺氧8h,测各组大鼠平均肺动脉压(mPAP)、肺动脉管壁面积(WA%)、肺动脉中膜厚度(PAMT)、右室肥厚指数(RVHI%);用原位杂交、免疫印迹和免疫组化法检测HIF1α、HIF2α和HIF3α基因表达。结果H7组mPAP为(18.40±0.40)mmHg(1mmHg=0.133kPa),H0组为(14.40±0.40)mmHg,H14组为(21.20±0.20)mmHg,H7组与H0组、H14组比较差异有统计学意义(P均<0.05);血管形态学显示,H7组WA%、PAMT、RVHI%分别为(47.8±0.8)%、(12.3±0.5)μm、(24.0±1.0)%,H14组分别为(60.3±0.4)%、(15.0±0.3)μm、(25.0±1.8)%,H0组分别为(35.5±1.3)%、(11.9±0.6)μm、(23.6±0.5)%,H21组分别为(65.0±0.7)%、(23.0±0.8)μm、(27.7±1.0)%,WA%H7组与H0组、H14组与H0组、H21组与H14组间比较差异均有统计学意义(P均<0.05)。原位杂交显示,H14组HIF1α、HIF2α、HIF3αmRNA水平的吸光度(A)值分别为0.200±0.020、0.080±0.010、0.170±0.010;H7组分别为0.050±0.020、0.160±0.020、0.160±0.020;H0组分别为0.050±0.010、0.140±0.020、0.060±0.010;H14组与H0组三者、H7组与H0组仅HIF3α比较差异有统计学意义(P均<0.05)。免疫组化表明,H3组HIF1α,HIF2α和HIF3α蛋白表达分别为0.200±0.020、0.020±0.010、0.050±0.010;H14组分别为0.160±0.010、0.100±0.020、0.160±0.010;H7组分别为0.220±0.020、0.030±0.010、0.180±0.020;H0组分别为0.050±0.010、0.020±0.010、0.040±0.010,H3组HIF1α蛋白、H14组HIF2α蛋白、H7组、H3组HIF3α蛋白分别与H0组比较差异有统计学意义(P均<0.05)。H7组免疫印迹显示在肺组织中HIF3α表达较H0组显著减弱,H3组HIF2α、HIF3α蛋白表达较H0组增强,随着缺氧时间延长,H14组较H7组更明显。结论3种HIFα表达差异可能在缺氧性肺动脉高压发病中发挥作用。
OBJECTIVE: To investigate the gene expression profiles of three hypoxia inducible factor α (HIFαα), HIF2α (HIF3α) in the pulmonary vascular wall during hypoxic pulmonary hypertension in rats. Methods Forty male Wistar rats were randomly divided into three groups: H0 group, H3 group, H3 group, H7 group, H14 group and H21 group Group), 8 in each group. The mean pulmonary arterial pressure (mPAP), pulmonary wall area (WA%), pulmonary arterial wall thickness (PAMT), right ventricular hypertrophy index ( RVHI%). The expression of HIF1α, HIF2α and HIF3α gene were detected by in situ hybridization, Western blotting and immunohistochemistry. Results The levels of mPAP in H7 group were (18.40 ± 0.40) mmHg (1.40 ± 0.40) mmHg in H0 group and (21.20 ± 0.20) mmHg in H14 group. The difference was statistically significant between H7 group and H0 group (P <0.05). The morphology of blood vessels showed that the percentages of WA%, PAMT and RVHI in H7 group were (47.8 ± 0.8)%, (12.3 ± 0.5) μm and (24.0 ± 1.0)%, respectively (35.3 ± 1.3)%, (11.9 ± 0.6) μm and (23.6 ± 0.5)% respectively in H0 group, (60.3 ± 0.4)%, (15.0 ± 0.3) μm and (25.0 ± 1.8) (P <0.05). Compared with H0 group, H14 group and H0 group, H21 group and H14 group, the difference was statistically significant (P <0.05) All <0.05). In situ hybridization showed that the absorbance (A) values of HIF1α, HIF2α and HIF3α mRNA in H14 group were 0.200 ± 0.020, 0.080 ± 0.010 and 0.170 ± 0.010, respectively. The H7 group was 0.050 ± 0.020, 0.160 ± 0.020 and 0.160 ± 0.020, respectively. H0 (0.050 ± 0.010,0.140 ± 0.020,0.060 ± 0.010, respectively). There was significant difference in HIF3α between H14 group and H0 group (all P <0.05). Immunohistochemistry showed that the protein expressions of HIF1α, HIF2α and HIF3α in H3 group were 0.200 ± 0.020, 0.020 ± 0.010 and 0.050 ± 0.010, respectively; those in H14 group were 0.160 ± 0.010, 0.100 ± 0.020 and 0.160 ± 0.010, respectively; those in H7 group were 0.220 ± 0.020 ± 0.010 and ± 0.080 ± 0.020 in H0 group, respectively. HIF1α protein in H0 group, HIF1α protein in H14 group, HIF3α protein in H7 group and H3 group were 0.050 ± 0.010, 0.020 ± 0.010, 0.040 ± 0.010, respectively Statistical significance (P <0.05). The expression of HIF3α in H7 group was significantly lower than that in H0 group in H7 group. The expression of HIF2α and HIF3α protein in H3 group was higher than that in H0 group. With the prolonged hypoxia time, H14 group was more obvious than H7 group. Conclusion The differences of the three HIFα expressions may play roles in the pathogenesis of hypoxic pulmonary hypertension.