HIF-2α对高原红细胞增多症模型大鼠骨髓CD71^＋细胞GATA-1表达的影响

Influence of HIF-2u on the expression of GATA-1 in bone marrow CD71^＋ cell of high altitude polycythemia rat model

目的探讨低氧诱导因子-2α（HIF-2α）对高原红细胞增多症（HAPC）模型大鼠骨髓CD71^＋细胞红系特异性转录因子GATA-1表达的影响。方法选用雄性SD大鼠48只，随机分为低海拔对照组（海拔2250m饲养）和HAPC模型组（海拔4300m饲养）。在海拔4300m自然环境下复制HAPC大鼠模型并采用骨髓细胞分类计数、血液学参数和血清红细胞生成素（EPO）检测进行验证。应用密度梯度离心和免疫磁珠分选相结合的方法分选大鼠骨髓CD71^＋细胞，Q—PCR和Westernblot法检测其HIF-2α、GATA-1 mRNA和蛋白的表达。低氧培养CD71^＋细胞，以最佳干扰序列HIF-2α shRNAi3转染96h，Q—PCR和Western blot检测HIF-2α、GATA-1 mRNA和蛋白的表达水平。结果骨髓细胞分类计数、血液学参数和血清EPO含量测定结果提示HAPC大鼠模型复制成功。HAPC模型组骨髓CD71^＋细胞HIF-2α、GATA-1 mRNA和蛋白的表达高于低海拔对照组，且HIF-2α与GATA-1在mRNA和蛋白表达均呈正相关（r=0．923，P〈0．01；r=0．838，P〈0．01）。HIF-2α shRNAi3干扰HAPC模型组骨髓CD71^＋细胞96h后，HIF-2α、GATA-1 mRNA和蛋白的表达均低于空白对照组和阴性对照组。结论HIF-2α对GATA-1表达的影响可能与HAPC的发生发展相关。

Objective To explore the influence of hypoxia-inducible factor-2 alpha （HIF-2α） on the expression of erythroid-specific transcription factor GATA-1 in bone marrow CD71^＋ cells of rat model with high altitude polycythemia （HAPC）. Methods A total of 48 male SD rats were selected and randomly divided into normal control group and HAPC group. HAPC model was established at an altitude of 4 300 meters in the natural environment and verified by bone marrow cell classification and counting, hematologic parameters and serum EPO detection. Bone marrow CD71^＋ cells were separated by a combination of methods with density gradient centrifugation and magnetic activated cell sorting. The changes of expression level of HIF-2α, GATA- 1 mRNA and proteins were detected by Q-PCR and Western blot. CD71^＋ cells were cultured under hypoxia condition and transfected with selected optimal HIF-2α shRNAi3 for 96 h. And the expression level of HIF-2α and GATA- 1 mRNA and proteins were detected by Q-PCR and Western blot. Results The results of bone marrow cell counts, the hematologic parameters and the serum EPO content showed that the HAPC rat model was successfully established. The expression of HIF-2α and GATA-1 mRNA and protein in bone marrow CD71^＋ cells of HAPC group was higher than that in control group （P〈0.05）. And HIF-2α and GATA- 1 of HAPC group were positively correlated at the expression levels of mRNA and protein, respectively （r=0.923, P〈0.01; r=0.838, P〈0.01）. However, the expression of HIF-2α and GATA-1 mRNA and protein in HAPC group was significantly lower than that in control groups after interfered by HIF-2α shRNAi3 for 96 h （P〈0.05）. Conclusion The effect of HIF-2α on GATA-1 expression may be correlated with the pathogenesis of HAPC.