汉防己甲素对人眼Tenon囊成纤维细胞表达细胞凋亡蛋白和转化生长因子的影响

Effect of tetrandrine eye drops on expression of apoptotic protein and transforming growth factor-132 in human Tenon capsule fibroblast in vitro

背景汉防己甲素（Tet）对体外培养的人眼Tenon囊成纤维细胞（TCFs）有抑制效应，但其作用机制尚不完全明确。目的观察Tet对体外培养的人眼TCFs中bax、bel-2、转化生长因子β2（TGF—β2）蛋白表达的影响，探讨其可能的作用机制。方法收集健康供体眼球巩膜组织，用组织块培养法培养、分离TCFs，并进行传代，将第3代细胞以1×10^5个／ml的密度接种于培养板，采用光学显微镜和免疫组织化学法对培养细胞进行鉴定。培养24h后，用培养液稀释Tet至浓度为1×10^-5mol／L，并加入培养孔中，对照组用1640培养液，继续培养24h后冲洗固定，采用TUNEL法检测细胞的凋亡情况；采用免疫组织化学法检测bax、bcl-2、TGF—β2蛋白在TCFs中的表达。结果培养的细胞形态学呈现成纤维细胞的特征，vimentin染色呈阳性反应。Tet组出现大量的TUNEL阳性细胞，而对照组TCFs未见明显凋亡细胞核出现。免疫组织化学检测表明，Tet组TCFs中bax、bcl-2、TGFβ2蛋白的表达率（A值）分别为0．577±0．009、0．430±0．012、0．341±0．017，对照组分别为0．320±0．015、0．819±0．021、0．624±0．014，Tet组bax的表达明显升高，bel-2和TGFβ2的表达明显下降，差异均有统计学意义（t=33．277、-35．356、-28．093，P〈0．01）。结论Tet通过上调人眼Tenon囊成纤维细胞中bax的表达，下调bcl-2、TGF-β2的表达，从而抑制TCFs的增生。

Background Our previous study determined that tetrandrine （Tet） has an inhibitory effect on the proliferation of human Tenon capsule fibrohlasts （TCFs） in vitro, hut its mechanism is poorly understood. Objective This study was to investigate the effect mechanism of Tet on human TCFs. Methods Human TCFs were isolated and cultured from scleral tissue of donor using explant technique. The cells were identified by vimentin antibody staining and morphology. The third generation of ceils were seeded in the culture plate at the density of 1 ×105 cells/ml. Twenty-four hours after inoculation,the Tet of 1× 10-5 mol/L was added in the well of culture plate, and the cells cultured only in 1640 medium served as the control group. The apoptosis of the cells was assessed by TUNEL,and the expressions of bax, bcl-2, transforming growth factor-β2 （TGF-β2 ） in TCFs were detected using immunochemistry. Results The cultured ceils showed the features of the fibroblasts in shape with the positive response for vimentin. A number of TUNEL positive cells were seen in Tet group and no TUNEL positive response was found in control group.