nmhaFGF和haFGF对放线菌素D诱导的人视网膜色素上皮细胞凋亡的保护作用

Protective effects of nmhaFGF and haFGF on apoptosis of hRPE cells induced by actinomycin D

目的探讨非促分裂型人酸性成纤维细胞生长因子(non-mitogenic human acidic fibroblast growth factor,nmhaFGF)和haFGF对放线菌素D诱导的人视网膜色素上皮(human retinal pigment epithelium,hRPE)细胞凋亡的保护作用,同时探讨2种浓度放线菌素D诱导hRPE细胞凋亡模型的复制条件。方法用0.25mg·L-1和0.50mg·L-1放线菌素D诱导hRPE细胞凋亡,用琼脂糖凝胶电泳检测2种浓度放线菌素D诱导hRPE细胞DNA断裂情况,并用流式细胞仪检测放线菌素D诱导hRPE细胞的凋亡率,确定诱导hRPE细胞凋亡的条件,分别以0.02mg·L-1、0.06mg·L-1、0.18mg·L-1、0.54mg.L-1和1.62mg·L-1的nmhaFGF和haFGF预作用于hRPE细胞24h,并设置正常细胞组和凋亡模型细胞组,观察nmhaFGF和haFGF对hRPE细胞凋亡的作用。结果0.25mg·L-1和0.50mg·L-1放线菌素D诱导hRPE细胞凋亡16h后细胞凋亡率分别为32.68%和53.27%,琼脂糖凝胶电泳结果显示0.25mg·L-1放线菌素D诱导细胞凋亡条带清晰,而0.50mg·L-1放线菌素D诱导hRPE细胞凋亡条带模糊,因此0.25mg·L-1放线菌素D作用hRPE细胞16h是诱导hRPE细胞凋亡的最佳条件。在nmhaFGF各剂量组中,随着nmhaFGF浓度升高,hRPE细胞凋亡率明显降低,其中1.62mg·L-1剂量组细胞凋亡率最低,为(11.49±0.50)%;相同浓度haFGF各剂量组细胞凋亡率也呈现不同程度的降低,但随着haFGF剂量升高,凋亡率下降的趋势不如nmhaFGF剂量组明显,在haFGF剂量组中,1.62mg·L-1剂量组细胞凋亡率最低,为(14.35±1.02)%,nmhaFGF和haFGF对hRPE细胞凋亡率的下降作用经比较差异无统计学意义。结论haFGF和nmhaFGF都可以发挥对hRPE细胞凋亡的保护作用。该保护作用可能是通过发挥其非促分裂活性实现的。

Objective To investigate the protective effects of non-mitogenic human acidic fibroblast growth factor （nmhaFGF） on apoptosis of human retinal pigment epithelium（hRPE） cells induced by actinomycin D, and to fred the best condition to create hRPE cells apoptotic models. Methods Apoptosis of hRPE cells was induced by 0.25 mg·L^-1 and 0.50 mg ·L^-1 actinomycin D, respectively. Agarose gel electrophoresis and flow cytometry were respectively used to detect the DNA fragmentation and apoptotic rates of hRPE cells induced by two different concentrations of actinomycin D to determine the apoptotic condition of hRPE cells apoptotic models. HRPE cells were pretreated with 0.02 mg ·L^-1 ,0.06 mg·L^-1 ,0.18 mg·L^-1 ,0.54 mg·L^-1 and 1.52 mg·L^-1 nmhaFGF and haFGF, respectively, for 24 hours before actinomycin D was dropped in. The control group and the model group were both set up. The effects of nmhaFGF and haFGF on apoptosis of hRPE cells were observed. Results The apoptotic rates of hRPE cells were 32.68% and 53.27% induced by0.25 mg·L^-1 and 0.50 mg·L^-1 actinomycin D for 15 hours, respectively. The strap of apoptotic hRPE cells induced by 0.25 mg ·L^-1 actinomycin D was more clear than that of 0.50 mg·L^-1 actinomycin D with agarose gel electrophoresis. So 0.25 mg ·L^-1 actinomycin D treating hRPE cells for 15 hours was the best condition to make the apoptotic model. With the increasing concentration of nmhaFGF, the apoptotic rate of hRPE cells decreased obviously, and the lowest apoptotic rate was （ 11.49 ± 0.50 ） % with 1.52 mg·L^-1 nmhaFGF. The decreasing tendency of apoptotic rate with increasing concentration for haFGF was not so obvious as that for nmhaFGF. The lowest apoptotic rate was （ 14.35 ± 1.02） % with 1.52 mg ·L^-1 haFGF. There was no statistical significance in reducing apoptotic rate of hRPE cells between the same concentrations of nmhaFGF and haFGF. Conclusion Both nmhaFGF and haFGF can protect the hRPE cells through reducing apoptotic rate, which may be associated with their non-mitogenic properties.