橙皮苷对人翼状胬肉成纤维细胞增生的抑制作用

目的　评价橙皮苷对体外培养人翼状胬肉成纤维细胞（human pterygium fibroblasts，HPF）增生的抑制作用，对照观察丝裂霉素C（mitomycin C，MMC）对HPF的影响，寻找治疗及预防翼状胬肉复发的中医中药方法。方法　将人翼状胬肉新鲜组织剪碎后进行体外贴壁细胞常规培养及传代，并采用波形蛋白（Vimentin）免疫细胞化学染色及DAPI染色对HPF进行鉴定。用24 μmol·L^-1、48 μmol·L^-1、64 μmol·L^-1、72 μmol·L^-1、96 μmol·L^-1、120 μmol·L^-1橙皮苷及1.5 μmol·L^-1、7.5 μmol·L^-1、30.0 μmol·L^-1 MMC作用于第3代HPF，24 h、48 h、72 h后采用MTT法检测细胞的增生抑制率，并筛选合适的浓度梯度及时间进行细胞凋亡检测，采用流式细胞仪检测细胞凋亡率。结果　根据MTT法检测细胞增生抑制率的结果，选用72 μmol·L^-1及48 μmol·L^-1 的橙皮苷作为实验组，将7.5 μmol·L^-1 MMC作为MMC标准对照组，无处理细胞作为空白对照组，培养48 h后流式细胞仪检测HPF的凋亡率。结果显示，72 μmol·L^-1橙皮苷组、48 μmol·L^-1橙皮苷组、7.5 μmol·L^-1 MMC标准对照组及空白对照组的细胞凋亡率分别为（41.10±1.04）%、（24.97±3.70）%、（48.60±6.94）%及（9.20±4.67）%，各组间HPF凋亡率整体存在差异（F=43.581，P<0.001），两两比较结果显示，除72 μmol·L^-1橙皮苷组与MMC标准对照组间差异无统计学意义（P>0.05）外，其余组间两两相比差异均有统计学意义（均为P<0.05）。结论　72 μmol·L^-1橙皮苷在抑制HPF生长方面有着与MMC等同的效果，主要是通过促进HPF凋亡进而抑制HPF的增生。

Inhibitory effects of hesperidin on proliferation of human pterygium fibroblasts

Objective To evaluate the inhibitory effect of hesperidin on proliferation of human pterygium fibroblasts(HPF)cultured in vitro,and observe the effect of mitomycin C(MMC)on HPF,in order to find the traditional Chinese medicine methods for the treatment and prevention of pterygium recurrence.Methods The fresh tissues of human pterygium were cut up,and routine culture and passage of adherent cells were made in vitro.HPF was identified by vimentin immunocytochemical staining and DAPI staining.The third generation HPF cells were treated with hesperidin(24μmol·L^-1,48μmol·L^-1,64μmol·L^-1,72μmol·L^-1,96μmol·L^-1,120μmol·L^-1)and MMC(1.5μmol·L^-1,7.5μmol·L^-1 and 30μmol·L^-1)for 24 h,48 h and 72 h.Cell proliferation inhibition rate was detected by MTT assay,and the appropriate concentration gradient and time were selected for detection of apoptosis.The apoptosis rate was measured by flow cytometry.Results According to the inhibition rate of cell proliferation detected by MTT assay,hesperidin at 72μmol·L^-1 and 48μmol·L^-1 were used as the experimental group,7.5μmol·L^-1 MMC as the standard control group,and untreated cells as the blank control group.The apoptosis of HPF was detected by flow cytometry,after cells being cultured for 48 h.The results showed that the apoptosis rates were(41.10±1.04)%,(24.97±3.70)%,(48.60±6.94)%and(9.20±4.67)%in 72μmol·L^-1 and 48μmol·L^-1 hesperidin groups,7.5μmol·L^-1 MMC standard control group and blank control group,respectively.There was difference in apoptosis rate of HPF among groups(F=43.581,P<0.001),and comparison between any two groups showed that there was no statistically significant difference between the 72μmol·L^-1 glucoside group and the 7.5μmol·L^-1 MMC group(P>0.05),but the differences between other pairs were statistically significant(all P<0.05).Conclusion 72μmol·L^-1 hesperidin has the same effect as MMC in inhibiting the growth of pterygium HPF,mainly by promoting the apoptosis of HPF and then inhibiting the proliferation of HPF.