氯离子通道-3反义寡核苷酸对内皮素-1促增殖体外培养的牛角膜内皮细胞的抑制作用

Inhibitive effects of chlorine channel 3 antisense oligonucleotides on endothelin-1 induced proliferation of cultured bovine corneal endothelial cells in vitro

目的观察氯离子通道-3(chloride channel3,ClC-3)反义寡核苷酸(antisense oli-gonucleotides,AS-ODN)对内皮素1(endothelin-1,ET-1)促增殖体外培养的牛角膜内皮细胞(bovine corneal endothelial cells,BCECs)的抑制作用。方法 BCECs细胞悬液接种培养12h后,通过脂质体介导转染不同浓度的ClC-3寡核苷酸。体外培养BCECs24h后加入200pmol.L-1ET-1,继续培养48h。通过倒置荧光显微镜观察BCECs寡核苷酸的摄取。采用逆转录聚合酶链反应、免疫印迹法检测ClC-3mRNA和蛋白的表达。采用MTT法观察BCECs增殖状况,计算细胞存活率,同时应用流式细胞仪检测细胞周期时相的变化,计算细胞增殖指数。结果寡核苷酸可被培养的BCECs摄取。ClC-3mRNA与蛋白的表达变化呈平行关系。与不加时相比,ClC-3AS-ODN浓度依赖性地减少BCECsClC-3mRNA和蛋白的表达,同样也浓度依赖性地降低BCECs MTT吸光度值(OD值)和细胞存活率。经100mg·mL-1ClC-3AS-ODN处理后,与对照组相比,细胞G0/G1期细胞比例明显增高,S期及G2期细胞比例则明显降低,出现明显的凋亡峰,增殖指数明显下降。结论 ClC-3AS-ODN可能通过减少ClC-3mRNA和蛋白的表达,浓度依赖性抑制ET-1对体外培养BCECs的增殖作用,并使细胞周期停滞在G1期。

Objective To investigate the inhibitive effects of chlorine channel 3（CIC-3）antisense oligonucleotides（AS-ODN）on endothelin-1（ET-1）induced proliferation of cultured bovine corneal endothelial cells（BCECs）in vitro.Methods After inoculated culturing for 12 hours in cell suspension,BCECs were mediated by liposome to transfer different concentrations of CIC-3 ODN.After being cultured for 24 hours in vitro,BCECs were cultured for another 48 hours with adding 200 pmol·L-1 ET-1.The uptake of BCECs ODN was observed under fluorescence microscope.The expression of ClC-3 mRNA and protein were detected by reverse transcription-polymerase chain reaction and immunoblotting.The proliferation of BCECs was observed by MMT assay.Cell survival rate was calculated,phase change of cell cycle was analyzed by flow cytometry,and cell proliferation index was calculated.Results ODN could be uptaken by cultured BCECs.The changes of ClC-3 mRNA paralleled to the levels of ClC-3 protein expression.Compared with that without phase,CIC-3 AS-ODN could decrease expression of CIC-3 mRNA and protein in BCECs with a dose-dependent manner,and also decrease MTT absorbance（OD value）and cell survival rate of BCECs with a dose-dependent manner.Compared with control group,the cell ratio of G0 to G1 stage were obviously higher,while S stage and G2 stage was much lower,apparent apoptosis peak presented and proliferation index decreased significantly after treatment of 100 mg·L-1 ClC-3 AS-ODN.Conclusion ClC-3 AS-ODN may decrease expression of ClC-3 mRNA and protein,inhibit ET-1 induced proliferation of BCECs with a dose-dependent manner and stop the cell proliferation at G1 stage.