Caspase-3对5-ALA诱导鳞状细胞癌Colo-16细胞凋亡效应的影响

Effects of caspase 3 on apoptosis of squamous-cell carcinoma Colo-16 cells induced by 5-ALA-PDT

目的探讨半胱氨酸天冬氨酸蛋白酶3(Caspase-3)对光敏剂5-氨基酮戊酸(5-ALA)诱导鳞状细胞癌Colo-16细胞凋亡效应的影响。方法取对数生长期的Colo-16细胞用于实验,将细胞分为对照组、5-ALA-光动力疗法(photodynamic therapy,PDT)组和Caspase-3抑制剂组(Z-DEVD-FMK),对照组不给予光敏剂和照光处理,5-ALA-PDT组给予0.4 mg/ml的5-ALA避光孵育4 h后,采用(功率密度10 m W/cm2,能量密度2.5 J/cm2)激光照射3 min,之后继续培养12 h。Caspase-3抑制剂组除孵育时同时加入Z-DEVD-FMK(终浓度40μmol),其余处理与5-ALA-PDT组相同。实验后收集各组细胞,通过免疫荧光观察各组细胞胞内Caspase-3荧光强度的变化,通过流式细胞术检测活性Caspase-3阳性细胞百分率和细胞凋亡率。结果免疫荧光结果发现对照组和Caspase-3抑制剂组Colo-16细胞胞浆中有微弱绿色荧光,表明Caspase-3表达量低,而5-ALA-PDT组中绿色荧光显著增强,表明其胞浆内Caspase-3含量显著增高。流式细胞仪的检测结果显示,对照组、5-ALA-PDT组和Caspase-3抑制剂组active-Caspase-3阳性细胞百分率分别为(2.26±0.16)%、(32.16±1.31)%和(3.36±0.31)%,5-ALA-PDT组高于对照组和Caspase-3抑制剂组,差异有统计学差异(P<0.05)。对照组、5-ALA-PDT组和Caspase-3抑制剂组的凋亡率分别为(2.35±0.22)%、(31.55±3.68)%和(11.46±2.25)%,三组之间两两比较,差异都有统计学意义(P<0.01)。结论 5-ALA-PDT可以诱导Colo-16细胞发生凋亡,且这种效应与caspase-3的激活密切相关。

Objective To investigate the role of caspase-3 in the apoptosis of squamous-cell carcinoma Colo-16 cells induced by 5-ALA-PDT. Methods Colo-16 cells in the logarithmic growth phase were divided into three groups： control group, 5-ALA photo- dynamic therapy （PDT） group, and caspase-3 inhibitor group （Z-DEVD-FMK）. Control group was not given photosensitizer or light treatment. 5-ALA PDT group was exposed to 3-minute laser irradiation at a power density of 10 mW/cm2 and energy density of 2.5 J/ cm2 after 4 hours of dark incubation with O. 4 mg/ml 5-ALA before being cultured for 12 hours. Caspase3 inhibitor group was treated in the same way as 5-ALA PDT group except that Z-DEVD-FMK at a final concentration of 40 μmol was added during incubation. Cells in each group were collected after experiment, the changes of intracellular caspase-3 fluorescence intensity were observed by immunofluorescence, and the percentage of positive cells and apoptosis rate of active caspase-3 were detected by flow cytometry. Results Immu- nofluorescence results showed that there was weak green fluorescence in the cytoplasm of Colo-16 cells in control group and Caspase-3 inhibitor group, indicating a low expression of caspase-3, while green fluorescence was significantly enhanced in 5-ALA-PDT group, showing a significant increase of caspase-3 content in cytoplasm of Colo-16 cells. Flow cytometry results showed that the respective percentage of active caspase-3 positive cells was （2.26 ± O. 16 ） %, （ 32.16 ± 1.31 ） % and （ 3.36 ± 0. 31 ） % in control group, 5-ALA PDT goup and caspase-3 inhibitor group respectively. The difference was statistically significant （ P 〈 0.05 ）. The apoptosis rate of control group, 5-ALA PDT group and caspase-3 inhibitor group was （2.35 ± 0. 22） %, （ 31.55 ± 3.68） % and （ 11.46 ± 2.25 ） %, respectively. Paired comparison was made between the three groups, and the differences were statistically significant （ P 〈 0.01 ）. Conclusions The apoptosis of Colo-16 cells induced by 5-ALA-PDT is closely related to the activation of caspase-3.