摘要
背景研究表明，羟基喜树碱（HCPT）和依托泊苷（VP-16）均能诱导人Tenon囊成纤维细胞（HTFs）的凋亡，但这两种药物联合应用对体外培养的HTFs是否有协同作用尚不明确。目的观察HCPT与VP-16联合应用后在促进体外培养的HTFs凋亡方面是否有协同性，并探讨其作用机制。方法人眼Tenon囊成纤维组织取自江苏省人民医院眼库，采用组织块培养法培养和传代HTFs，采用免疫组织化学法检测HTFs中波形蛋白的表达。第4代HTFs接种于96孔板，分别将不同质量浓度的HCPT（1、5、10、50、100mg／L）、VP-16（0．6、2．5、5．0、25．0、50．0mg／L）、HCPT＋VP-16（质量比为2：1，终质量浓度分别为0．80、3．75、7．50、37．50、75．00mg／L）加入细胞培养孔处理24h，用CCK-8法检测各组药物对细胞增生的抑制率。将HTFs分为空白对照组、HCPT（50mg／L）处理组、VP-16（25mg／L）处理组、HCPT＋VP-16（37．5mg／L）处理组，药物处理24h后用流式细胞仪检测各组细胞的凋亡率。提取细胞内总蛋白，采用Westernblot法检测各药物处理组细胞内caspase-3、活化型caspase-3、bax、bcl-2、JNK、p-JNK、Akt、p-Akt的表达水平。结果培养的细胞呈多边形或不规则形，波形蛋白表达阳性。不同质量浓度HCPT、VP-16和HCPT＋VP-16作用24h后，随着药物质量浓度的增加，对HTFs增生的抑制率增加，差异均有统计学意义（HCPT：F=41．34，P=0．00；VP-16：F=62．60，P=0．00；HCPT＋VP-16：F=46．77，P=0．00），HCPT处理组、VP-16处理组和HCPT＋VP-16处理组药物的半数抑制浓度（IC50）分别为80．99、27．93、19．81mg／L，HCPT与VP-16联合应用的合并指数（cI）为0．399，表明VP-16和HCPT具有强协同作用。空白对照组、HCPT处理组、VP-16处理组和HCPT＋VP-16处理组HTFs的凋亡率分别为（4．87±0．78）％、（11．20±1．94）％、（12．67±1．51）％和（19．77±2．01）％，差异有统计学意义（F=18．23，P〈0．01），其中HCPT处理组、VP-16处理组和HCPT＋VP-16处理组HTFs凋亡率较空白对照组明显升高，差异均有统计学意义（q’=15．67、16．32、26．88，P〈0．01）。Westernblot法检测结果表明，与空白对照组相比，HCPT处理组、VP-16处理组和HCPT＋VP-16处理组HTFs中活化型easpase．3、bax、p-JNK表达的灰度值明显增加，差异均有统计学意义（P〈0．01），且HCPT＋VP-16处理组HTFs中上述各因子表达的灰度值较HCPT处理组、VP-16处理组明显增加，差异均有统计学意义（P〈0．01），而easpase-3、JNK、Akt的表达无明显变化。与空白对照组比较，bcl-2、p-Akt在HCPT处理组、VP-16处理组和HCPT＋VP-16处理组HTFs中的表达量明显下降，HCPT＋VP-16处理组较HCPT处理组、VP-16处理组的表达量明显减少，差异均有统计学意义（P〈0．01）。结论单独使用HCPT、VP-16均能诱导体外培养的HTFs凋亡，其作用均呈剂量依赖性，HCPT与VP-16联合应用有强协同作用。HTFs中p-JNK、bax的表达上调和p-Akt、bcl-2表达的下调参与联合用药引起的协同效应。
Background Previous study showed that both hydroxycamptothecin （HCPT） and etoposide （VP-16） can induce the apoptosis of human Tenon capsule fibroblasts （HTFs）. However, whether the combination of HCPT with VP-16 enhance the efficacy of drugs is unknown. Objective This study was to investigate the synergistic effect and its mechanism of HCPT combined with VP-16 on apoptosis of HTFs. Methods Human Tenon capsule tissue was obtained from the eye bank of Jiangsu Province People＇s Hospital. HTFs were cultured in vitro using explant method and identified by immunofluorescence with vimentin. The fourth generation of cells were incubated in 96-well plate, and different concentrations of HCPT （ 1,5,10,50,100 mg/L） , VP-16 （ 0. 6,2.5,5.0, 25.0,50.0 mg/L） and HCPT＋VP-16 （2 ： 1 ,final concentrations O. 80,3.75,7.50,37.50,75.00 mg/L） were added for 24 hours. The inhibiting rate of drugs to HTFs growth was detected using CCK-8 kit. The HTFs were divided into blank control group, HCPT （50 mg/L） treated group, VP-16 （25 mg/L） treated group and HCPT ＋ VP-16 （37.5 mg/L） treated group, and the apoptosis rates of HTFs in various groups were assayed by flow cytometry. The expressions of caspase-3, cleaved caspase-3, bax, bcl-2, JNK, p-JNK, Akt, p-Akt in the cells were detected by Western blot assay. Results Cultured cells grew well with the polygon shape and positive response for vimentin. The inhibiting rate was elevated with the increase of drug dosage 24 hours after addition of drugs （HCPT： F=41.34,P= 0.00 ;VP-16 ： F = 62.60, P = O. 00 ; HCPT ＋ VP-16 ： F = 46.77, P = O. 00 ）. The half maximal inhibitory concentrations （ ICs0 ） of HCPT, VP-16, HCPT＋VP-16 were 80.99,27.93,19.81 mg/L, respectively, and the combined index （CI） of HCPT with VP-16 was 0. 399, showing a stronger synergistic action. The apoptotic rates of HTFs were （4.87± 0.78） %,（11.20±1.94）%,（12.67±1.51）% and （19.77±2.01）% in the blank control group,HCPT treated group,VP-16 treated group and HCPT＋VP-16 treated group, respectively, with a significant difference among them （F= 18.23, P〈 0. 01 ）, and the apoptotic rate was significantly raised in the HCPT ＋ VP-16 treated group, HCPT treated group and VP-16 treated group compared with the blank control group （ q＇= 15.67,16.32,26.88, all at P〈 0. 01 ）. Compared with the blank control group,the grey scale values of cleaved caspase-3 ,bax,p-JNK in the cells of HCPT＋VP-16 treated group, HCPT treated group and VP-16 treated group were significantly increased （all at P〈 0. 01 ） ,and those in the HCPT＋VP-16 treated group significant ascent in comparison with the HCPT treated group and VP-16 treated group （ all at P〈0. 01 ）. However, the changes of caspase-3, JNK and Akt expression were insignificant. The grey scale values of bcl-2 and p-Akt in the HTFs of the HCPT,VP-16 and HCPT＋VP-16 treated groups were significantly lower than those of the blank control group,with a dominant reducing in the HCPT＋VP-16 treated group （all at P〈0. 01 ）. Conclusions HCPT and VP-16 induce the apoptosis of HTFs in vitro at a dose-dependent manner. The combination of HCPT with VP-16 has a stronger synergistic efficacy. The up-regulation of p-JNK and bax as well as the down-regulation of p-Akt and bcl-2 in HTFs are involved in the coaction of HCPT and VP-16.
出处
《中华实验眼科杂志》
CAS
CSCD
北大核心
2014年第2期125-130,共6页
Chinese Journal Of Experimental Ophthalmology
基金
国家自然科学基金资助项目（81271001）
