氯离子通道阻滞剂对人眼小梁细胞增生及凋亡的影响

Effect of a chloride channel inhibitor on proliferation and apoptosis of human trabecular meshwork cell

背景氯离子通道阻滞剂5-硝基-2-（3-苯丙胺）苯甲酸（NPPB）对家兔心肌细胞缺血-再灌注损伤时的细胞凋亡有促进作用，而小梁细胞上也存在C1C型氯离子通道及容积敏感性氯离子通道，但NPPB究竟会对小梁细胞的形态和功能产生什么影响尚不清楚。目的探讨氯离子通道阻滞剂NPPB对人眼小梁细胞增生及细胞周期、细胞凋亡的影响。方法将处于对数生长期的永生株人眼小梁细胞进行培养并以1×10^6个／ml的密度接种于96孔培养板，将不同浓度（10、50、100μmol／L）的NPPB分别加入小梁细胞培养基中，通过四甲基偶氮唑蓝（MTT）法检测各组小梁细胞的增生情况以吸光度（A）值表示；采用流式细胞术测定小梁细胞的细胞周期。将100mg／L5-氟尿嘧啶（5-Fu）加入培养的细胞中，而另-组培养的细胞中加入100mg／L5-FU＋100μmol／LNPPB，用AnnexinV—PI法检测各组小梁细胞的凋亡情况；罗丹明123法检测细胞线粒体膜电位（Adam），分别评估各浓度NPPB对培养的人小梁细胞生长和存活的影响。结果3种不同浓度的NPPB与小梁细胞共同孵育48h后4个组小梁细胞的增生率差异有统计学意义（F=7．230，P=0．006），50Izmol／L、100Fxmol／LNPPB作用后小梁细胞的A值明显低于10μmol／LNPPB组，50μmol／LNPPB组、100μmol／LNPPB组与空白对照组比较差异均有统计学意义（t=1．610，P=0．025；t=12．270，P=0．001）。小梁细胞培养基中加入NPPB48h后，G0／G1期的细胞比例增多，S期细胞比例减少，各细胞周期小梁细胞的比例与未加NPPB的组比较，差异均有统计学意义（P〈0．05）。用5-FU作用24h及48h后，100mg／L5-Fu组和100mg／L5-FU＋100μmol／LNPPB组的细胞凋亡率均较其各自空白对照组增加（t24h=2．130，P=0．023；t48h=4．810，P=0．011）；100mg／L5-Fu＋100μmol／LNPPB组细胞凋亡率明显高于100mg／L5-Fu组，差异均有统计学意义（t24h=1．980，P=0．037；t48h=1．290，P=0．028），小梁细胞线粒体膜电位降低，差异均有统计学意义（t24h=1．580，P=0．029；F48h=6．200，P=0．015）。结论氯离子通道阻滞剂NPPB可抑制小梁细胞增生，抑制小梁细胞通过G1／S期限制点进入s期；NPPB对5-Fu诱导的小梁细胞凋亡有促进作用，与内源性线粒体凋亡通路相关。

Background 5-Nitro-2-（ 3-styrene-acrylic amine） benzoic acid （NPPB）, a chloride channel inhibitor,has a promoting effect on cell apoptosis in myocardial ischemia and reperfusion of domestic rabbit. The CIC chloride channel has been found in the ocular trabecular cells. However,the effect of NPPB on the shape and function of trabecular cells is unclear. Objective This study was performed to investigate the effect of NPPB on the proliferation,cell cycle progression and apoptosis of human trabecular meshwork cells. Methods The immortalized human trabeular cell strain was cultured, and logarithmic-phase cells were incubated in 96-well plates at a density of 1×10^6/ml. Different concentrations of NPPB （10,50,100 μmol/L） were added to the medium, and the MTT assay was used to assess the growth and proliferation of the cells. Flow eytometry was used to evaluate the cell cycle. Then, 100 mg/L 5-FU or 100 mg/L 5-FU＋ 100 μmol/L NPPB was used to induce cell apoptosis, which was assessed by Annexin V-PI. The membrane potential of mitochondria was examined using rhodamine 123 （△ψm）. Results After 48 hours of treatment with NPPB, the abosorbency （A value） of the cells was gradually lowered with the increasing dose of NPPB,with significant differences among the 4 groups （F = 7. 230,P = 0. 006 ）. Compared with the 10 μmol/L NPPB group,the A values were significantly declined in the 50 and 100 μmol/L NPPB groups （t= 1. 610, P = 0. 025 ;t = 12. 270, P = 0. 001 ）. Forty-eight hours after exposure to NPPB, the percentage of cells in G0/G1 phasewas increased and that in the S phase was decreased. The percentages of cells in different phases of cell cycle were significantly different in comparison with their control groups （without NPPB ）（ P〈0. 05 ）. Twenty-four and 48 hours after the treatment with 100 mg/L 5-FU, the apoptosis rates of the cells were raised in the 100 mg/L 5-FU group and 100 mg/L 5-FU＋ 100 Ixmol/L NPPB group compared to the without NPPB group （t24h = 2. 130, P = 0.023; t4sh =4. 810,P=0. 011 ） ;while that in the 100 mg/L 5-FU＋100 μmol/L NPPB group was higher than the 100 mg/L 5-FU group （ t24 h = 1. 980, P = 0. 037 ; t48h = 1. 290, P = 0. 028 ） , and the mitochondrial membrane potential was lowered （ t24 h = 1. 580, P = O. 029 ; F48 h = 6. 200, P = O. 015 ）. Conclusions NPPB suppresses the proliferation of human trabecular cells and promotes the ceils to enter S phase via the G1/S check point. In addition,C1C might be involved in an anti-apoptosis mechanism through the internal mitochondrial pathway.