紫杉醇对人Tenon囊成纤维细胞增生的抑制作用及机制

Effect of paclitaxel on proliferation of human Tenon fibroblast and its probably mechanism

背景人Tenon囊成纤维细胞（HTFs）的异常增生是导致抗青光眼滤过手术失败的主要原因。寻找抑制HTFs增生的药物对抑制青光眼术后功能性滤过泡的瘢痕化有重要意义。目的观察紫杉醇对体外培养的HTFs增生、凋亡、细胞周期以及基质金属蛋白酶-1（MMP-1）表达的影响。方法收集抗青光眼滤过术中切除的人眼Tenon囊组织，用组织块培养法培养HTFs并进行传代，取3～6代细胞用于实验。采用小鼠抗人角蛋白抗体、小鼠抗人波形蛋白抗体、小鼠抗人结蛋白抗体、小鼠抗人S-100抗体行免疫组织化学染色鉴定培养的HTFs。在培养基中分别加入0、1×10^-8、1×10^-8、1×10^-6mol／L紫杉醇，采用实时细胞电子分析系统（RT—CES）观察不同浓度紫杉醇作用后HTFs的细胞指数变化；采用DAPI染色法观察各组细胞的凋亡情况；用流式细胞术检测不同浓度紫杉醇作用后HTFs各细胞周期比例；采用实时定量PCR（real—timePCR）和ELISA法检测各组HTFs中MMP-1mRNA和蛋白的表达量，分别以MMP-1mRNA／GAPDHmRNA和MMP-1蛋白质量浓度（μg／L）表示。结果组织块原代培养7d内可见细胞游出，呈长梭形和不规则三角形，培养的细胞抗波形蛋白抗体染色阳性，而抗角蛋白抗体、抗结蛋白抗体和抗S-100抗体染色阴性。培养基中加入紫杉醇作用24h后，1×10^-8、1×10^-7、1×10^-6mol／L紫杉醇组平均细胞指数值分别为1．09±0．191、0．665±0．093和0．473±0．117，均明显低于0mol／L紫杉醇组的1．514±0．283，差异均有统计学意义（均P=0．000）；紫杉醇作用后，HTFs的细胞核内可见呈DAPI蓝色荧光的颗粒状物质，荧光强度随着紫杉醇浓度的增加而增强。l×10^-8、1×10^-7、1×10^-6mol／L紫杉醇分别作用12h后，G，／M期HTFs的比例分别为（9．20±0．80）％、（12．37±0．45）％和（13．80±0．35）％，明显高于0mol／L紫杉醇组的（7．17±0．50）％，差异均有统计学意义（P=0．005、0．000、0．000）。与0mol／L紫杉醇组比较，1×10^-8、1×10^-7、1×10^-6mol／L紫杉醇作用30min后HTFs中MMP-1mRNA的相对表达量均明显下降，差异均有统计学意义（均P=0．000）；1×10^-8、1×10^-7、1×10^-6mol／L紫杉醇作用24h后，HTFs中MMP-1蛋白的表达量均明显低于0mol／L紫杉醇组，差异均有统计学意义（P=0．010、0．002、0．001）。结论1×10^-8～1×10^-6mol／L紫杉醇可抑制体外培养HTFs的增生，诱导细胞凋亡，阻断细胞的有丝分裂，其作用机制可能与下调细胞中MMP-1的表达有关。

Background The excessive growth of human Tenon fibroblasts （HTFs） is a primary cause of failure of anti-glaucomatous filtering surgery. To seek a drug of anti-fibrosis is of an important significance for improving the successful rate of anti-glaucomatous filtration surgery. Objective The goal of this study was to investigate the effect of paclitaxel on proliferation of HTFs in vitro. Methods Human Tenon tissue was obtained during the anti-glaucomatous filtering surgery. HTFs were cultivated using explant method and 3-6 generations of cells were used in the experiment. The cells were identified by immunochemistry using keratin, vimentin, fibronectin and S-100. Different concentrations （0,11×10^-8、1×10^-7、1×10^-6mol/L） of paclitaxel were added into the medium, and then the cell indexes （CI） in the various groups were detected by real-time cell electronic sensing （RT-CES） 24 hours after affection of paclitaxel. Apoptosis of the cells was examined using DAPI staining, and the proportion of the ceils in different cycles were assayed by flow cytometer 12 hours after addition of paclitaxel. The expressions of matrix metalloproteinase-1 （ MMP-1 ） protein and mRNA were detected by ELISA and real-time PCR,respectively. Results The cells migrated from the cultivated tissue within 7 days with the fihrocyte-like shape. The cells showed the positive response for vimentin and absent response for keratin, fibronectin and S-100. The CI values were 1. 093 ± 0. 191,0. 665±0. 093 and 0. 473 ±0. 117 in the 1×10^-8、1×10^-7 and 1×10^-6 mol/L paclitaxel groups, showing significant rise in comparison with the 1. 514±0. 283 of the 0 mol/L paclitaxel group （all at P= 0. 000）. The cell nuclei were normal in the 0 mol/L paelitaxel group, however, blue-fluorescent particles and apoptotic bodied were found in the cell nuclei after affection of paclitaxel. The proportion of G2/M phase of cells were （9.20±0. 80） % , （12.37±0.45）% and （13.80±0.35）% in the 1×10-8 mol/L,1×10-7 mol/L and 1×10-6 mol/L paclitaxel groups, which were higher than the （7.17±0.50） % in the 0 mol/L paclitaxel group （P=0.005,0.000,0.000）. In addition,the relative expressing level of MMP-1 mRNA （ MMP-1 mRNA/GAPDH mRNA） and the expression level of MMP-1 protein in the HTFs were significantly lower in the 1 × 10^-8 mol/L, 1×10^-7 mol/L and 1×10^-6 mol/L paclitaxel groups than those in the 0 mol/L group （ all at P〈0.05 ）. Conclusions Paclitaxel at the concentrations of 1× 10^-8 mol/L-1×10^-6 mol/L inhibits the proliferation of HTFs in vitro by arresting the cellular mitosis and inducing cell apoptosis. These effects probably associated with down-regulation of MMP-1 expression in the HTFs.