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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 observe the effect of hypoxia on the proliferation of bovine retinal pigment epithelium (RPE) cells and the expression of bcl-2 gene. Methods MTT method and MTT method were used to culture RPE cells The cells were seeded in 24-well plates and the density of each well was 5 × 10 4. The cells were divided into 4 groups: group A was cultured in hypoxic environment (3% O2, 5% CO2, 92% N2, 37 ℃) Group C: The supernatant of cells cultured in hypoxia environment for 24 hours was added to the culture of bovine RPE cells cultured in normal environment; Group D: Normal cells were cultured under normal conditions (5% CO2, 95% air, 37 ℃) Cultured for 24 h, the cells were cultured in normal cultured RPE cells. 2 4h after removal of the line MTT colorimetric and anti-bcl-2 protein antibody staining. Results Compared with group B, group C and group D, the former MTT absorbance (A) [formerly OD] was higher than the latter (P <0.05); the anti-bcl- The antibody positive rates of group A and group B were 72.60% and 38.64% respectively. The positive rates of group C and D were 83.2% and 2.80% respectively. Positive staining in the cytoplasm, especially near the nuclear membrane stain deep, some were fine granular. Conclusion Hypoxia can promote the proliferation of bovine RPE cells and the expression of bcl-2.