| 摘要 目的 观察氯离子通道-3（chloride channel 3 ，ClC-3）反义寡核苷酸（antisense oligonucleotides,AS-ODN）在内皮素1(endothelin-1， ET-1)对体外培养牛角膜内皮细胞（bovine corneal endothelium cells， BCECs）增殖和ClC-3表达的影响中的作用。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寡核苷酸于体外培养BCECs，作用24h后加入200 pM ET-1，继续培养48h。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。采用四甲基偶氮唑盐比色分析法（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BCECs摄取。ClC-3 mRNA及其蛋白的表达结果变化呈平行关系。与对照组相比，ClC-3 AS-ODN浓度依赖性地减少BCECs ClC-3 mRNA和蛋白的表达。同时ClC-3 AS-ODN浓度依赖性地降低BCECs MTT吸光度值（OD值）和细胞存活率。经100µg/ml ClC-3 AS-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-3 AS-ODN可能通过减少ClC-3 mRNA和蛋白的表达，浓度依赖性抑制ET-1对培养BCECs增殖作用，并使细胞周期停滞在G1期。Abstract Objective To study the effect of chlorine channel 3(ClC-3) antisense oligonucleotides(AS-ODN) on proliferation and ClC-3 expression of endothelin-1(ET-1) induced cultured bovine corneal endothelium cells(BCECs). Methods 12h after seeded, cultured BCECs in vitro were transferred by ClC-3 oligonucleotides through liposome for 24h, then added to 200 pmol/l ET-1 and after 48h, cells were collected to prepare experiments. The uptake of ClC-3 oligonucleotides was observed by fluorescence microscope. The expression of ClC-3 mRNA was detected in cultured BCECs by reverse transcription-polymerase chain reaction (RT-PCR). ClC-3 protein was performed with human anti- ClC-3 polyclonal antibody by Western blot. MMT assay was used to examine the effect on proliferation of cells, meanwhile phase change of cell cycle was analyzed by flow cytometry(FACS). Results The uptake of ClC-3 AS-ODN was observed by cultured BCECs transiently transfected with oligonucleotides specific to ClC-3. The changes of ClC-3 mRNA ran paralleled to the levels of ClC-3 protein expression. Transient transfection of BCECs with ClC-3 AS-ODN caused an inhibition effect on expression of ClC-3mRNA and its protein of BCECs with ET-1 treated in the concentration dependent pattern, whereas sense and missense oligonucleotides resulted in no effects on ClC-3 mRNA and protein expression. Meanwhile ClC-3 AS-ODN decreased MTT absorbance value(OD value) and cell survival rate of BCECs in a dose-dependent manner. Compared with control group, after treatment of 100µg/ml ClC-3 AS-ODN, the cell ratio in G1 stage was obviously higher while S stage and G2 stage was much lower, apparent apoptosis peak presented, and proliferation index decreased significantly. Conclusion ClC-3 AS-ODN may decrease ClC-3 mRNA and protein expression, and then inhibit on ET-1–induced cell proliferation of BCECs in a dose-dependent manner and the cell proliferation was stagnant at G1 stage. These results strongly suggest that ClC-3 may be the Cl_ channel involved in BCECs proliferation. |
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