PDGF抗体对体外培养人视网膜色素上皮细胞增生的抑制作用

Inhibition of anti-PDGF on proliferation of cultured human retinal pigment epithelial cells in vitro

目的:探讨血小板源性生长因子(PDGF)抗体对体外培养人视网膜色素上皮细胞(hRPE)增生的影响。方法:体外培养hRPE分为对照组和实验组,对照组未加入抗体,实验组分别加入1×10-6、5×10-6、1×10-5、5×10-5和1×10-4mg.L-1PDGF抗体。采用生长曲线法及MTT比色法检测PDGF抗体对hRPE细胞增殖的影响,并用流式细胞术检测细胞周期的变化,透射电镜观察抗体作用后细胞超微结构的变化。结果:1×10-6mg.L-1PDGF抗体对hRPE细胞有轻度促增生作用,MTT结果显示其抑制率为-11.74%。5×10-6、1×10-5、5×10-5和1×10-4mg.L-1PDGF抗体对细胞有抑制作用,作用72 h抑制率分别为12.55%、43.72%、55.10%和55.10%。台盼蓝染色检测细胞存活率,各组细胞活力均大于80%。流式细胞仪检测结果显示,5×10-5mg.L-1PDGF抗体作用72 h,细胞明显阻滞于G2/M期,可见凋亡指数明显增加。透射电镜结果显示,5×10-5mg.L-1PDGF抗体作用72 h电镜下可见胞质空化,核染色质轻度趋边凝聚,有早期凋亡的改变。结论:PDGF抗体对hRPE细胞的增生有明显的抑制作用,在一定范围内随着抗体浓度的增大对hRPE细胞增生的抑制作用逐渐加强。PDGF抗体对hRPE细胞增生的抑制作用主要是依赖于诱导细胞的凋亡,而不是促使细胞变性坏死,作用性质比较温和。

Objective To study the inhibition of anti-PDGF on proliferation of cultured human retinal pigment epithelial cells （hRPE） in vitro. Methods hRPE were cultivated and were exposed to different concentrations of antiPDGF （0, 1×10^- 6, 5×10^-6, 1×10^- 5, 5×10 ^-5 and 1×10^ 4 mg. L^- 1） respectively. Growth curves were measured with cell counting and the vitalities of cells were examined by percentage of vital cells and total cells. Using MTT staining colorimetric to measure the inhibitory rate. The changes of cell cycle of hRPE were collected and their growth were detected with FCM analysis and the morphological changes of cells were observed by light microscope and electron microscope. Results Anti-PDGF of 1 × 10^- 6 mg· L^-1 stimulated hRPE proliferation slightly. Anti-PDGF at dosages ranging from 5 × 10 ^-6 mg· L ^-1 to 1 × 10^-4 mg·L^- 1 inhibited cell proliferation effectively in a dose-dependent and time-dependent manner （P〈0.05）. The inhibitory rates of anti-PDGF were 12.55%, 43.72%, 55.1% and 55.1%. hRPE might be blocked at G2/M phase in their growth cycles after treated with anti-PDGF （5 × 10^-5 mg· L^- 1 ） for 72 h, and the electron microscopy result showed a significant reduction of microvilli, margination of nuclear ehromatin and intact plasmalemma. Conclusion Anti-PDGF can inhibit hRPE in a dose-dependent and time-dependent manner. It＇s inhibitory function lies in inducing apoptosis without resulting in cell putrescence.