芹黄素对人眼Tenon囊成纤维细胞增生的影响

Inhibitory effect of apigenin on human Tenon capsule fibroblasts

背景青光眼滤过手术后人Tenon囊成纤维细胞（HTFs）的增生是滤过手术失败的主要原因，寻找抑制术后HTFs增生的药物可提高滤过手术的成功率。目的观察芹黄素对HTFs体外生长的抑制效应并探讨其作用机制。方法斜视手术中获取的人眼Tenon囊组织剪成1mm×1mm×1mm的组织块，进行原代培养，免疫荧光法进行细胞波形蛋白检测以鉴定培养的HTFs。取第3～5代HTFs接种于96孑L板中，加入0、20、40、80、160μmol／L芹黄素后继续培养，于24、48、72h后分别取一块96孔板，在酶联免疫检测仪上检测560nm波长处各组细胞的吸光度（A。）值，磺基罗丹明B（SRB）法测定细胞的增生能力。分别在培养基中加入40、80、160μmol／L芹黄素，同时在不同浓度芹黄素组培养基中均加入10汕g／LBrdU，继续培养48h，在各组同一个视野下分别拍摄荧光显微镜和光学显微镜照片，计算BrdU的标记率，以未添加任何芹黄素（0μmol／L）组为对照组。用Hoechst33258染色后在荧光显微镜下观察培养细胞的形态学改变，采用流式细胞技术检测细胞的凋亡情况和细胞周期的变化。结果培养的细胞生长状态良好，免疫荧光法检测可见细胞对波形蛋白呈阳性反应，为细胞质中均匀的绿色荧光，确定培养的细胞为HTFs。SRB法检测结果显示，HTFs的增生值（A。）随着芹黄素浓度的增加而逐渐下降，同浓度芹黄素组HTFsA。值随着作用时间的延长逐渐下降，差异均有统计学意义（F㈣u：480．306，P=0．000；F目目=555．144，P=0．000）。0、40、80~mol／L芹黄素作用HTFs48h后BrdU的标记率分别为（87．860＋-0．632）％、（61．520＋-4．306）％、（23．480~4．472）％，各组之间的总体比较差异有统计学意义（F=299．347，P=0．000），其中40μmol／L、80μmol／L芹黄素组HTFsBrdU的标记率均明显低于0μmol／L芹黄素组，差异均有统计学意义（P〈0．05）。Hoechse33258染色发现，不同浓度芹黄素组的细胞数目减少，随着芹黄素浓度的增加，细胞核固缩和变形的细胞数目增加。流式细胞术检测发现，随着芹黄素浓度的增加，G。／G，期细胞的百分数逐渐增加，而S期和G：／M期细胞的百分数逐渐下降，差异均有统计学意义（Fc㈣1=58．621，P=0．000；Fs=32．357，P=0．001；Fc2／M=83．998，P=0．000）。0、40、80、160~mol／L芹黄素作用72h后，细胞的凋亡率分别为（4．77±0．21）％、（13．24±1．35）％、（18．33±1．86）％、（31．58±2．77）％，4个组的总体差异有统计学意义（F=204．791，P〈0．05）。结论芹黄素通过诱导细胞凋亡，并将细胞阻滞于C／C期而抽柏JHTFs的士曾堆苴椎田里剂骨和时问俯觞幛，

Background Proliferation of the human Tenon capsule fibroblasts （HTFs） is a main cause of failure of filtering surgery. To search the drug of inhibiting the growth of the HTFs is essential for the improvement of successful rate of filtering surgery. Objective The aim of this study was to investigate the inhibitory effect of apigenin on HTFs and its mechanism. Methods Human Tenon capsular tissue was obtained during the strabismus correction surgery. HTFs was primarily cultured using explant method and identified using vimentin by immunochemistry. The 3-5 generation of cells were incubated to 96-well plate. Apigenin of 0,20,40,80,160 p, mol/Lwas added into the medium,respectively,for 24,48,72 hours, and the proliferation of HTFs was detected by sulfonyl chloride （SRB） at the wavelength of 560 nm （A560 ）. Bromodeoxyuridine （BrdU） of 10 μg/L was added to culture the eells for 48 hours to calculate the labeling rate of BrdU. The morphology of the cells was observed using Hoechst 33258 staining,and apoptosis and cells eyele were evaluated by flow eytometry. Results Cultured eells grew well with the positive response for vimentin,showing the green fluoreseence in cytoplasm. SRB assay showed that the A560 value was gradually deelined with the increase of the dosage of apigenin and prolong of time （ Fgroup = 480. 306, P = 0. 000 ;Fime = 555. 144 ,P = 0. 000 ）. The labeling rate after 0,40,80μmol/L apigenin aeted for 48 hours was （87. 860 ＋0. 632）% , （61. 520-＋4. 306 ）% and （23. 480-＋4. 472 ）% , showing a significant difference among the three groups （ F = 299. 347,P-- 0. 000）. The labeling rate of HTFs for BrdU was significantly decreased in the 40 and 80μmol/L apigenin groups compared with the 0 μmol/L apigenin group （P〈0.05）. Hoeehse 33258 staining found that the number of the HTFs was gradually decreased and the cell number of karyopyknosis and nuclear deformation was inereased with the inerease of apigenin dosage. Percentage of cells in Go/G1 phase were raised and that in S and Gz/M phase were declined in the higher dosage apigenin group, with a significant difference among the different groups （Fc0/ci =58. 621 ,P=0. 000;Fs =32. 357 ,P=0. 001 ;Fcz/M =83. 998,P=0. 000）. In the 72nd hour after acted by 0, 40,80,160μmol/L apigenin,the apoptosis rate of HTFs was （4.77＋0.21） %, （13.24-＋1.35）%, （18.33-＋ 1.86）% ,（31.58 ＋2.77 ）%, respectively, with a statistieally significant difference among the four groups （F = 204. 791 ,P〈0.05）. Conclusions Apigenin restrains the growth of HTFs by evoking G0/GI eell eyele arrest and indueing apoptosis in a dosage- and time-dependent manner.