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目的探讨肺腺癌细胞缺氧后胞红蛋白表达变化的特点与规律。方法将人肺腺癌细胞株A549扩增后,分为4组:常氧组(对照组),缺氧4、12、24h组。对照组置常规培养箱(5%CO2、95%空气,37℃)中培养,各缺氧组置缺氧培养箱中培养(2%O2,5%CO2,93%N2,37℃)不同时间(4、12、24h)后,取肺腺癌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肺腺癌细胞的细胞浆,缺氧各组胞红蛋白染色强度均高于常氧组。免疫印迹灰度分析结果显示缺氧各组胞红蛋白表达量高于常氧组(153·5±7·1、233·3±26·8、276·2±41·4vs95·7±13·1,P<0·05)。而缺氧12、24h组胞红蛋白表达高于缺氧4h组,差异有统计学意义(P<0·05)。缺氧4、12、24h组mRNA水平均显著高于常氧组(85·6±15·4、136·2±27·4、148·8±20·1vs56·8±18·5,P<0·05),缺氧12、24h组较缺氧4h时组更进一步增高(P<0·05)。结论胞红蛋白是分布于肺腺癌细胞的细胞浆中的一种珠蛋白。肺腺癌细胞缺氧4h后胞红蛋白表达上调,胞红蛋白表达增加可能是细胞对缺氧肿瘤的一种代偿反应。
Objective To investigate the characteristics and rules of the changes of hemoglobin in lung adenocarcinoma cells after hypoxia. Methods Human lung adenocarcinoma cell line A549 was amplified and divided into 4 groups: normoxia group (control group), hypoxia group 4,12,24h. The control group was cultured in a conventional incubator (5% CO2, 95% air, 37 ° C), and cultured in anoxic incubator (2% O2, 5% CO2, 93% N2, 37 ° C) (4, 12, 24 h), the lung adenocarcinoma cells were harvested for immunohistochemical staining and Western blot analysis of the changes of protein distribution and expression. The changes of hemoglobin mRNA levels were analyzed by reverse transcription polymerase chain reaction (RT-PCR). Results Immunohistochemical staining showed that hemoglobin was mainly distributed in the cytoplasm of lung adenocarcinoma cells. The staining intensity of hemoglobin in hypoxia groups was higher than that in normoxia group. Immunoblotting results showed that the expression of hemoglobin in hypoxia group was higher than that in normoxia group (153.5 ± 7 · 1,233 · 3 ± 26 · 8,276 · 2 ± 41 · 4vs95 · 7 ± 13 · 1, P <0 · 05). However, the expression of CRP in hypoxia group was higher than that in hypoxia group at 12 and 24 hours, the difference was statistically significant (P <0.05). The levels of mRNA in hypoxia group at 4,12,24h were significantly higher than those in normoxia group (85.6 ± 15.4,136.2 ± 27.4, 148.8 ± 20.1vs56.8 ± 18.5, P < 0 · 05), hypoxia 12,24 h group was further increased compared with 4 h hypoxia group (P <0 · 05). Conclusion Hemoglobin is a globin distributed in the cytoplasm of lung adenocarcinoma cells. Lung adenocarcinoma cells after 4h hypoxia increased the expression of hemoglobin, increased expression of hemoglobin may be a compensatory response of cells to hypoxia tumors.