缺氧对牛视网膜色素上皮细胞增生和凋亡基因bcl-2表达的影响

The effect of hypoxia on proliferation of cultured bovine retinal pigment epithelial cells and expression of the antiapoptotic protein bcl-2

目的 观察缺氧对培养的牛视网膜色素上皮 (retinal pigment epithelium,RPE)细胞增生和凋亡基因 bcl- 2表达的影响。 方法 用四甲基偶氮唑蓝 [3- (4,5 - dimethylthiazole- 2 yl) - 2 ,5 - diphenyl te-trazolium bromid,MTT]比色法和免疫组织化学的方法 ,将培养的 RPE细胞接种于 2 4孔板 ,每孔细胞密度为 5× 10 4,分成 4组 :A组置入缺氧环境中培养 (3% O2 ,5 % CO2 ,92 % N2 ,37℃ ) ;B组置入正常环境中培养 (5 % CO2 ,95 %空气 ,37℃ ) ;C组 :将缺氧环境培养 2 4h的细胞上清液加到正常环境培养的牛 RPE细胞中培养 ;D组 :将正常环境培养 2 4h的细胞上清液加到正常环境培养的牛 RPE细胞中培养。2 4h后分别取出行 MTT比色法及抗 bcl- 2蛋白抗体染色。 结果 A组与 B组、C组与 D组比较 ,前者细胞 MTT实验吸光度 (A)值 [旧称光密度 (OD) ]均高于后者 (P<0 .0 5 ) ;抗 bcl- 2蛋白抗体染色 ,A、B组阳性率分别为72 .6 0 %、38.6 4% ;C、D组阳性率分别为 83.2 0 %、2 1.80 %。阳性染色在细胞浆 ,近核膜处染色尤深 ,部分呈细颗粒状。 结论 缺氧促进牛 RPE细胞增生及凋亡基因 bcl- 2表达。

Objective To study the effect of hypoxia on proliferation of cultured bovine retinal pigment epithelium (RPE) cells and expression of the antiapoptotic protein bcl-2. Methods The bovine RPE cells were cultured under normal and hypoxic chamber respectively. After 24 hours, the proliferation of RPE cells was evaluated by[3-(4,5-dimethylthiazole-2yl)-2,5-diphenyl tetrazolium bromide, MTT]-test. At the same time, anti-bcl-2 protein antibody was examined by immuno-histochemistry method. Results The A value in the hypoxia group was higher than that in the normal group after 24 hours (P<0.05 )in MTT-test. Positive staining for anti-bcl-2 protein antibody was seen in 72.6% cells in hypoxia group and 38.64% in normal group. The positive staining was more obvious near the nucleus, and fine granules scattered in cytoplasm of some cells. Conclusion Hypoxia can stimulate the proliferation of RPE cells and expression of antiapoptotic protein bcl-2. The results indicate that bcl-2 may play an important role in mediating the proliferation activity of RPE cells.