摘要
目的探讨人视网膜色素上皮(RPE)细胞中整合素α5表达的调节机制。方法人RPE细胞原代、传代培养,RT-PCR及流式细胞术观察血清组、PD98059组和对照组整合素α5 mRNA和蛋白的表达,Western blot法检测各组人RPE细胞中MAPK磷酸化水平。结果与对照组相比,血清组能促进整合素α5 mRNA和蛋白的表达,而PD98059(20μmol/L)组可抑制此促进作用;流式细胞仅显示培养24h后3组整合素α5荧光强度分别为1.94±0.22,4.56±0.25,2.39±0.14,差异有统计学意义(P<0.05)。Western blot结果显示30min后血清组ERK1/2磷酸化激活水平最高,PD98059组抑制ERK1/2的磷酸化激活,对照组ERK1/2的磷酸化激活作用很弱。结论10%小牛血清能促进人RPE细胞表达整合素α5,ERK1/2的磷酸化激活参与此过程。
Objective Proliferation of retinal pigment epithelium （ RPE ） cell plays a key role in pathogenesis of proliferative vitreoretinopathy（PVR）, lntegrin α5 is involved in this process. This study presented a regulatory mechanism of integrin α5 expression in RPE stimulated by serum in vitro. Methods Human RPE cells were primarily cultured in vitro,and the 3rd to 6th generations of cells were used in this study. Cultivated cells were assigned to 0. 1% calf serum ＋ DMEM group （control group） ,20 μmol/L PD98059 ＋ 0. 1% calf serum ＋ DMEM group（PD98059 group）and 10% calf serum ＋ DMEM group （serum group）. The expressions of mRNA and protein of integrin α5 in different groups were detected by RT-PCR and flow cytometry, and Western blot was used to observe phosphorylation level of mitogen-activated protein kinase（MAPK）. Results Passaged cells showed the spindle-like and irregular in shape with a brown-yellow staining in cytoplasm for panytokeratin. The integrin α5 expression level in human RPE cells was increased in serum group in comparison with control group and PD98059 group（P 〈 0.01 ）,and the expression of integrin α5 was lowest in PD98059 group. The fluorescence intensity of integrin α5 was 4.56 ± 0. 25,1.94 ± 0. 22 and 2.39 ± 0. 14 respectively after cultivation with PD98059 or serum for 24 hours, indicating a significant difference between them （ F = 260. 258 ,P 〈 0. 05 ）. The phosphorylation level of MAPK was highest in the serum group and lowest in PD98059 group, The phosphorylation of MAPK in control group was low after treatment with PD98059 or serum for 30 minutes. Conclusion 10% calf serum can promote the expression of integrin α5 in human RPE, and phosphorylation of MAPK may play a role in this process.
出处
《眼科研究》
CAS
CSCD
北大核心
2008年第2期121-124,共4页
Chinese Ophthalmic Research
