培养心肌细胞缺氧时胞红蛋白表达变化特点

Hypoxia upregulates cytoglobin in rat cardiac myocytes

目的探讨心肌细胞缺氧后胞红蛋白表达变化的特点与规律。方法利用酶消化法及差速贴壁法分离培养新生2—3d大鼠的心肌细胞，分为4组：对照组，缺氧4、12、24h组。对照组置常规培养箱（5％CO，、95％空气，37℃）中培养，缺氧组置缺氧培养箱中培养（2％O2，5％CO2，93％N2，37℃），于不同时间（4、12、24h）后，取心肌细胞进行免疫组化染色和免疫印迹分析蛋白水平的分布与表达的变化，用逆转录PCR法分析胞红蛋白mRNA水平的变化。结果免疫组织化学染色显示胞红蛋白主要分布于细胞质，缺氧各组胞红蛋白染色强度均高于常氧组（对照组）。免疫印迹灰度分析结果显示，缺氧4h组胞红蛋白表达量高于常氧组，但差异无统计学意义（P〉0．05），而缺氧12、24h组胞红蛋白表达高于常氧组，差异有统计学意义（P〈0．05）。缺氧4、12、24h组胞红蛋白mRNA水平均高于常氧组（P〈0．05），缺氧12、24h组较缺氧4h组更进一步增高（P〈0．05）。结论胞红蛋白分布于心肌细胞的细胞质。培养心肌细胞缺氧4h后胞红蛋白转录增强，缺氧12h后蛋白水平增强。胞红蛋白表达增加可能是心肌细胞对缺氧的一种代偿反应。

Objective To explore the characteristics of cytoglobin expression in cardiac myocytes after hypoxia. Methods The cardiac myocytes from neonatal Wistar rats were exposed to hypoxia （2% 02, 5% CO2, 93%N2, 37 ℃ ） for4, 12, 24 h, and the control cells exposed to normoxia （95% air, 5%CO2,37 ℃ ）. The expression of cytoglobin protein cardiac myoeytes was assayed by immunohistochemical staining and Western blotting, while the mRNA level by reverse transcription PCR. Results Cytoglobin located in plasma of cardiac myocytes. As compared to normoxic cardiac myocytes, immunohistochemical staining showed cytoglobin protein in all hypoxic cardiac myocytes was enhanced, and Western blotting showed the cytoglobin of the cells exposed to hypoxia for 4 h was of no significance （ P 〉 0.05 ） and that for 12 and 24 h was of statistical significance （P 〈0.05）. The cytoglobin mRNA level of hypoxic cardiac myocytes was higher than normoxic cells （P 〈0.05）. The cytoglobin expression of cells exposed to hypoxia for 12 and 24 h was higher than for 4 h （P 〈 0.05）. Conclusion Cytoglobin is a plasma protein in cardiac myocytes. Hypoxia can upregulate the expression of cytoglobin in cardiac myocytes.