| 目的 研究内皮素-1（endothelin-1, ET-1）对体外培养牛角膜内皮细胞（bovine corneal endothelium cells, BCECs）增殖的影响并探讨氯离子通道-3（chloride channel 3，CLC-3）在其中的作用。
方法 采用免疫组化染色法检测增殖细胞核抗原(proliferating cell nuclear antigen, PCNA) 的表达，MTT法检测细胞增殖，同时应用流式细胞仪检测细胞周期变化。应用逆转录聚合酶链反应（RT-PCR）、免疫印迹（Western blot）方法检测CLC-3 mRNA和蛋白的表达。免疫荧光测定CLC-3蛋白的表达部位。采用反义核酸技术，观察CLC-3基因在ET-1对体外培养牛角膜内皮细胞影响中的作用。
结果 体外培养BCECs在转录和翻译水平均有内源性CLC-3基因表达，表达产物定位于细胞膜和部分细胞浆。ET-1剂量依赖性的增加培养的BCECs CLC-3 mRNA和蛋白的表达，促进其增殖（MTT OD值和PCNA表达阳性细胞平均吸光度值A增加），10 pmol/L时起作用，200 pmol/L时发挥最大作用。CLC-3反义寡核苷酸（antisense oligodeoxynucleotide，AS-ODN）可以剂量相关性的下调CLC-3 mRNA及蛋白的表达，浓度依赖性抑制200 pmol/L ET-1促进细胞增殖的作用，并使细胞周期停滞于G1期。
结论 ET-1通过开放氯通道，增加CLC-3 mRNA和蛋白的表达，促进培养的BCECs增殖。CLC-3 AS-ODN阻断CLC-3氯通道，抑制ET-1促进细胞增殖的作用，使细胞周期停滞于G1期。
The role of chloride channel-3 in the proliferation effect of endothelin-1 on bovine corneal endothelium cells
abstract Objective To find best culture methods for bovine corneal endothelium cells (BCECs) in vitro, and investigate the effect of endothelin-1(ET-1) on proliferation of cells and the role of chloride channel- 3 (CLC-3) in the process.
Methods The expression of proliferating cell nuclear antigen (PCNA) was oberserved by immunohischemistry staining, MTT assay was used to detect cell proliferation and meanwhile flow cytometry (FACS) was used to analyze cell cycle phase change. The expression of CLC-3 mRNA and CLC-3 protein were tested by reverse transcription-polymerase chain reaction (RT-PCR) and western blot methods. Immunofluorescence technique was used to localize the expression of CLC-3. Antisense oligonucleotide technique was used to observe the role of CLC-3 in the effect of ET-1 on BCECs.
Results The expression of endogeneous CLC-3 gene could be detected at the level of transcription and translation, and CLC-3 protein expression localized in cell membrane and part of cell plasma of cultured BCECs in vitro. ET-1 promoted cell proliferation(MTT OD value and average absorbance (A) of positive PCNA expression of cells increased) and presented dosage correlation at the rank from 10 pmol/L to 200 pmol/L. The expression levels of CLC-3 mRNA and CLC-3 protein increased with ET-1 in a dose dependent manner. Antisense oligodeoxynucleotide of CLC-3 (CLC-3 AS-ODN) decreased the expression of CLC-3 mRNA and protein, and had inhibition effect on 200 pmol/L ET-1–induced cell proliferation in a concentration dependent pattern, and resulted in the cell cycle was stagnant at G1 stage, prevented to entry cell proliferation phase.
Conclusion Through opening chloride channel, ET-1 increases the expression levels of CLC-3 mRNA and its protein, and promotes the proliferation of BCECs in vitro. By inhibiting CLC-3 chloride channel, CLC-3 AS-ODN has the inhibition effect on ET-1–induced cell proliferation and results in that cell cycle is stagnant at G1 stage.
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