汉防己甲素抑制人眼Tenon囊成纤维细胞的凋亡效应

Inhibitory effect of tetrandrine on the apoptosis of cultured human Tenon's capsule fibroblasts

目的研究汉防己甲素(Tet)抑制人眼Tenon囊成纤维细胞(TCFs)的凋亡效应。方法用组织块混合消化液培养法体外培养人眼TCFs,并对培养的传代细胞进行形态学鉴定。通过透射电镜、TUNEL法和流式细胞仪观察Tet抑制人眼TCFs的凋亡作用。结果人眼TCFs体外生长良好。透射电镜下见Tet处理后的人眼TCFs细胞质固缩,呈现凋亡表现;TUNEL法可见Tet组出现大量的阳性凋亡细胞;流式细胞仪检测TCFs经Tet作用后G0/G1期细胞减少22.2%,S期细胞增加20.53%,G2/M期细胞增加1.6%,Tet抑制TCFs细胞周期于G1期,Tet组凋亡率明显高于对照组(P=0.000)。结论Tet可以诱导人眼TCFs发生凋亡,进而发挥其抑制增生的作用。

Background Excessive conjunctival sear formation is a main cause of filtering surgical failure in glaucoma. It has been reported that the failure rate of filtering surgery is a tough problem in the research of glaucoma. Research showed that tetrandrine （Tet） suppress the proliferation of cultured human fibroblasts of Tenons capsule （TCFs） in vitro, but its possible mechanism is still unclear up to now. Objective The aim of this study is to investigate the inhibitory effects of Tet on the apoptosis of fibroblasts of Tenons capsule and mechanism. Methods The Tenon＇ s capsule was obtained from donor eyes of Zhongshan Eye Center Eye Bank for further use. Human Tenon＇ s capsule fibroblasts were cultured by explant culture method in mixed medium in vitro and the third to seventh generations of cells were collected for the experiment. The subcuhured cells were identified by morphology observation and Vimentin staining. The apoptosis of cultured fibroblasts in Tet-treated group and control group was studied by using TUNEL （TdT- mediated dUTP nick end ]abling,TUNEL）,flow cytometry （FCM） and transmission electron microscope. The cell number in different cellular cycle was calculated in Ted-treated group and control group. Results The cultured cells reached confluence in two weeks after cultured and presented the spindle or triangle shape with the radial-like or vortexiu-like arrangement . The cells showed the positive staining for Vimentin. Apoptosis changes of cultured cell and apoptosis bodies were seen under the transmission electron microscope. Apoptotic cell nuclei were observed in Tet group by TUNEL. FCM result showed that the cells at G0/G1 phase decreased by 22.2% ,and the cells at S and G2/M phases increased by 20. 53% and 1.6% in Ted-treated group. Significant differences in the numbers of cells in different cellular cycles and cell numbers of apoptosis were found between Tet-treated group and control group（ P = 0. 000）. Conclusion Tet can inhibit the proliferation of human TCFs by inducing apoptosis in vitro.