表皮生长因子对兔角膜上皮细胞增殖及细胞周期相关蛋白作用的研究

目的 探讨表皮生长因子(EGF)对兔角膜上皮细胞的增殖刺激作用及其与细胞周期相关蛋白表达的关系.方法 实验研究.原代培养的兔角膜上皮细胞,以不同浓度(0、15、10、25、50 ug/L)EGF作用72 h、以10ug/L EGF作用不同时间(0、2、4、6、8、10 d)后,通过相差显微镜观察细胞形态,并通过水溶性四氮唑(WST-1)细胞增殖实验检测细胞增殖情况;采用Western blot检测10ug/L EGF作用0、24、48、72 h后细胞周期蛋白D1、细胞周期蛋白依赖性激酶4(CDK4)及细胞周期蛋白依赖性激酶抑制蛋白(CDKIs)p21、p27及p18的表达状况.采用SPSS 11.5统计软件对各实验组结果 数据进行统计分析,两样本比较采用t检验,多个样本比较采用单因素方差分析.结果 比较不同浓度的EGF对兔角膜上皮细胞的增殖影响效果,加入5、10、25及50ug/L EGF组72 h的细胞增殖率分别为9.0%、23.5%、20.8%及17.7%,与未加入EGF(0 ug/L)的对照组增殖率相比较,差异有统计学意义(F=45.48,P<0.01),其中10 ug/L EGF对兔角膜上皮细胞的增殖促进作用最强,且相同浓度EGF组兔角膜上皮细胞的增殖率具有明显的时间依赖性.10 ug/L EGF作用0、24、48及72 h,各细胞周期相关蛋白表达量与B肌动蛋白表达量的比值:细胞周期蛋白D1为0.253、0.591、0.885、1.043,CDK4为0.422、0.588、0.804、1.241,表达水平随时间推移逐渐升高,而p27为0.225、0.163、0.107、0.082,表达水平随时间推移逐渐下降,p21为0.432、0.391、0.407、0.329,p18为0.268、0.274、0.231、0.309,表达不随时间推移而变化.结论 EGF对兔角膜上皮细胞具有明显的促增殖作用,可能与其上调细胞周期蛋白D1和CDK4以及抑制p27的表达密切相关.(中华眼科杂志,2009,45:153-157)

Effect of epidermal growth factor on expression of cell cycle-regulatory proteins and proliferation of rabbit corneal epithelial cells

Objective To investigate the effects of epidermal growth factor (EGF) on the proliferation of cultured rabbit corneal epithelial (RCE) cells and the expression of cell cycle-regulatory proteins in these cells. Methods It was an experimental study. Cultured RCE cells were exposed to EGF at different concentrations (0, 1,5, 10, 25 and 50 ug/L) and different time(0, 2, 4, 6, 8 and 10 days). Cell morphologic changes were observed under a phase contrast microscope. Cell proliferation was measured using the WST-1 cell proliferation assay. The expression of cell cycle-regulatory proteins, cyclinD1, CDK4, p27, p21 and p18, was examined using Western blot analysis. The data was statistically analyzed with SPSS 11.5 software, difference between two groups and multiple groups was compared by t test and ANOVA, respectively. Results Treatment with EGF did not obviously change the cell morphology. EGF significantly induced the proliferation of RCE cells in a dose-and time-dependent manner. In cells treated with different concentrations of EGF, the proliferation rate of 5, 10, 25 and 50 ug/L EGF group at 72 h were 9.0%, 23.5%, 20.8% and 17.7%, respectively. The difference was statistically significant as compared with the control group (F=45.48, P<0.01). EGF at 10 ug/L showed maximally stimulating effect. After EGF treatment for 0.24. 48 and 72 h. the expression of cyclinD1 and CDK4 proteins was markedly increased (cyclinD1/beta-actin ratio was 0.253, 0.591, 0.885 and 1.043; CDK4/beta-actin ratio was 0.422, 0.588, 0.804 and 1.241 at different periods, respectively). Furthermore, p27 protein expression was significantly inhibited by EGF (p27/beta-actin ratio was 0.225, 0.163, 0.107 and 0.082 after EGF treatment for 0, 24, 48 and 72 h, respectively). However, there was no detectable difference in p21 and p18 protein expression between EGF treatment and control groups (p21/beta-actin ratio was 0.432, 0.391, 0.407 and 0.329 and p18/beta-actin ratio was 0.268, 0.274, 0.231 and 0.309, respectively). Conclusions This study suggests that EGF could induce RCE cell proliferation. EGF up-regulates cyclinD1 and CDK4 and down-regulates p27 in RCE cells. These changes may be the causes for EGF-induced proliferation of RCE. (Chin J Ophthalmol, 2009,45:153-157)