人酸性成纤维细胞生长因子对过氧化氢诱导的人视网膜色素上皮细胞坏死的保护作用

Protective effects of human acidic fibroblast growth factor on necrotic hRPE cells induced by hydrogen peroxide

目的研究人酸性成纤维细胞生长因子(human acidic fibroblast growth factor,haFGF)对人视网膜色素上皮(human retinal pigment epithelium,hRPE)细胞的保护作用。方法用0.2mmol.L-1过氧化氢培养hRPE细胞36h制作hRPE细胞坏死模型为模型组,用1mg.L-1haFGF预培养hRPE细胞12h、24h、48h,并设置空白对照组,用台盼蓝染色在倒置显微镜下观察细胞坏死情况,用MTT法检测haFGF预作用hRPE细胞不同时间细胞的死亡率,并用8g.L-1琼脂糖凝胶电泳检测haFGF不同时间对hRPE细胞完整性的影响。结果模型组细胞死亡率为(36.97±2.81)%,空白对照组细胞死亡率为(1.05±0.04)%,haFGF预作用细胞12h、24h、48h细胞死亡率分别为(32.50±4.06)%、(19.77±3.68)%、(26.45±3.19)%。其中,模型组与空白对照组细胞死亡率相比、haFGF预作用24h与模型组相比差异均有显著统计学意义(均为P<0.01),而haFGF预作用12h、48h细胞死亡率与模型组相比差异均无统计学意义(均为P>0.05)。台盼蓝染色结果显示haFGF预作用细胞24h,hRPE蓝染细胞核数明显减少,而预作用12h染色细胞核数减少不显著,预作用48h蓝染细胞核数显著增加。琼脂糖凝胶电泳显示:模型组细胞DNA呈现"涂布"状,说明细胞坏死明显,haFGF预作用细胞24h"涂布"状基体消失,提示hRPE细胞DNA完整性尚好,而预作用12h和48hDNA"涂布"状仍明显存在。结论1mg.L-1的haFGF可以显著拮抗过氧化氢对hRPE细胞的损伤,预作用24h时haFGF对hRPE细胞的保护作用最佳。

Objective To investigate the protective effects of human acidic fibroblast growth factor（haFGF） on human retinal pigment epithelium（hRPE） cells.Methods The hRPE cells were cultured with 0.2 mmol·L^-1 hydrogen peroxide for 36 hours to induce necrotic cell models.The hRPE cells were pretreated with 1 mg·L^-1 haFGF for 12 hours,24 hours and 48 hours,respectively,and normal control group was set up.Cellular necrosis was evaluated with trypan blue staining under inverted microscope.The mortalities of hRPE cells were detected with MTT method at different times after pretreated with haFGF.Effects of haFGF on cell integrity of hRPE cells at different times were measured with 8 g·L^-1 agarose gel electrophoresis.Results The mortalities of hRPE cells were （36.97±2.81）%,（1.05±0.04）%,（32.50±4.06）%,（19.77±3.68）% and （26.45±3.19）% in necrotic cell model group,normal control group,groups after pretreated with haFGF for 12 hours,24 hours and 48 hours,respectively.There was significant difference in cell mortality between necrotic cell model group and normal control group,also between group after pretreated with haFGF for 24 hours and necrotic cell model group（both P〈0.01）.But no significant difference was found in either group after pretreated with haFGF for 12 hours or 48 hours compared with necrotic cell model group（both P〉0.05）.Trypan blue staining showed that number of blue stained nucleus was obviously decreased after pretreated with haFGF for 24 hours,but not significant after pretreated with haFGF for 12 hours or 48 hours.Agarose gel electrophoresis showed that cell DNA was with spreading shape in necrotic cell model group,indicating obvious cellular necrosis;The spreading shape was disappeared basically after pretreated with hRPE for 24 hours,indicating good integrity of hRPE cell DNA;While the spreading shape was still obvious after pretreated with haFGF for 12 hours or 48 hours.Conclusions 1 mg·L^-1 haFGF can obviously inhibit the hRPE injury induced by hydrogen dioxide.The best condition to protect hRPE cells is the time after pretreated with haFGF for 24 hours.