羟喜树碱诱导的人Tenon囊成纤维细胞凋亡及其机制

Hydroxycamptothecin-induced apoptosis of human Tenon capsule fibroblast and its mechanism

背景青光眼滤过手术后人Tenon囊成纤维细胞（HTFs）增生是滤过泡功能下降的主要原因，羟喜树碱是诱导肿瘤细胞及多种非肿瘤细胞凋亡的药物。目前已有羟喜树碱诱导成纤维细胞凋亡的相关报道，但其作用机制尚不完全清楚。目的探讨羟喜树碱是否能诱导HTFs凋亡并研究其作用机制。方法取离体〈6h的供体结膜囊组织，以组织块培养法原代培养HTFs，用含质量分数10％胎牛血清（FBS）的DMEM培养基继续传代培养，用波形蛋白及角蛋白对培养的细胞进行鉴定，取3～6代细胞进行实验。分别将0．Ol、0．05、0．10g／L羟喜树碱加入培养基中作用5min，未加入羟喜树碱的细胞作为对照组，采用细胞计数试剂盒-8（CCK8）法检测各组细胞的增生能力并进行比较。用0．10g／L羟喜树碱处理细胞，继续培养24h，用annexinV／PI双染流式细胞仪检测细胞凋亡率，用JC-1染色检测HTFs线粒体膜电位，采用Westernblot法检测HTFs中半胱氨酸天冬酶-3（caspase-3）、easpase-9及线粒体／细胞质中细胞色素C（cytC）蛋白的表达，对各组中的测量结果进行比较。结果0、0．01、0．05、0．10g／L羟喜树碱组的HTFs增生值（A450）分别为0．9716±0．0608、0．8035±0．0346、0．7048±0．0446和0．6265±0．0286，总体差异有统计学意义（F=26．372，P=0．002），0．01、0．05、0．10g／L羟喜树碱组HTFsA450值均低于对照组，差异均有统计学意义（P〈0．05），以0．10g／L羟喜树碱组HTFsA。，。值最低。AnnexinV／PI双染色检测发现，0．10g／L羟喜树碱组HTFs凋亡率为（18．72±1．41）％，明显高于对照组的（3．67±O．36）％，差异有统计学意义（t=-10．374，P=0．001）；0．10g／L羟喜树碱组HTFs中caspase-3、caspase-9蛋白的表达强度明显强于对照组。JC-1染色发现，0．10g／L羟喜树碱处理5min后细胞质中单体JC．1的绿色荧光明显强于对照组，而线粒体中聚合态JC-1的红色荧光弱于对照组。Westernblot检测发现，0．10g／L羟喜树碱组HTFs线粒体中cytC蛋白表达灰度值为0．0124±0．0016，高于对照组的0．0216±0．0096，而0．10g／L羟喜树碱组HTFs细胞质中eytC蛋白表达灰度值为0．0605±0．0022，低于对照组的0．0301±O．0016，差异均有统计学意义（线粒体：z=4．865，P：0．014；细胞质：t=-11．177，P=0．001）。结论羟喜树碱诱导HTFs凋亡．引起线粒体的稳态破坏．激活线粒体凋亡涂径。

Background The hyperplasia of human Tenon capsule fibroblasts （HTFs） is a common cause of filtering surgery failure in glaucomous eye. Researches demonstrated that hydroxycamptothecin is a cell cycle arresting drug and induce apoptosis of cancer and fibroblasts. However,its mechanism is currently less understood. Objective This study was to investigate whether hydroxycamptothecin induce the apoptosis of HTFs and explore the possible mechanism. Methods Human Tenon capsule tissue was obtained from EyeBank of Jiangsu Province Hospital. HTFs were cultured using explant method in vivo and passaged in DMEM containing 10% FBS. The cells were identified using vimentin and keratin by immunochemistry,and the cells of generation 3-6 in the logarithmic growth phase were used in the experiment. The cells were incubated with 0. 01,0.05 or 0. 10 g/L hydroxycamptothecin for 5 minutes respectively,and the cells without any hydroxycamptothecin were served as the control group. Cell viability then was assessed by cell counting kit-8 （CCK8） for the optimal inhibition concentration. The cells were treated by 0. 10 g/L hydroxycamptothecin for 24 hours, and the apoptotic rate of the cells were assayed with annexin V/PIdouble-staining. Mitochondrial membrane potential of HTFs was assessed using JC-1 staining. The expressions of caspase-3,caspase-9 and cytochrome C （cyt C） in mitochondria and cytoplasm of HTFs were detected by Western blot. Results The proliferative value （A450 ） of the HTFs 0,0.01,0.05,0. 10 g/L was 0. 9716＋0. 0608,0. 8035＋ 0. 0346,0. 7048-＋0. 0446,0. 6265-＋0. 0286, with a significant difference （F = 26. 372, P = 0. 002 ）. A450 of HTFs in the 0.01 , 0. 05,0. 10 g/L groups was significantly lower than the control group （P〈0.05） , with the lowest A450 value in the 0. 10 g/L group. The apoptotic percentage of HTFs was （18.72＋ 1.41 ）% , in the 0. 10 g/L hydroxycamptothecin group and that of the control group was （3.67-＋0.36）% , showing a significant difference between them （t =- 10. 374, P= 0. 001 ）. The expression intensity of caspase-3 and caspase-9 protein in HTFs was higher in the 0. 10 g/L hydroxycamptothecin group than that in the control group. JC-1 staining showed that the green fluorescence of the monomer JC-1 in cytoplasm was stronger in the 0. 10 g/L hydroxycamptothecin group than that in the control group, but the red fluorescence of the polymer JC-1 in the 0. 10 g/L hydroxycamptotheein group was weaker than that in the control group. The grey scale of eyt C protein in HTFs in mitochondrion was 0. 0605 _＋0. 0022 in the 0.10 g/L hydroxycamptothecin group,showing a significant increase in comparison with 0. 0301 -＋0. 0016 of the control group （t=4. 865,P = 0.014）. However, the grey scale of cyt C protein in cytoplasm was declined in the 0. 10 g/L hydroxycamptothecin group than that in the control group （ 0. 0605-＋0. 0022 vs. 0. 0301 -＋0. 0016 ） （ t = - 11. 177, P = 0.001 ）. Conclusions Hydroxycamptothecin can induce the apoptosis of HTFs through activating the mitochondrial apoptosis pathway.