| 摘要:目的 探讨体外培养牛角膜内皮细胞的最佳方法，并研究内皮素-1（ET-1）对其增殖和移行的影响及氯离子通道-3（chloride channel 3，CLC-3）在其中的作用。方法 采用2种培养方法和4种培养基体外培养牛角膜内皮细胞。通过损伤模型检测细胞移行。采用免疫组化染色法、MTT法和流式细胞仪检测PCNA表达、细胞增殖和细胞周期变化。免疫荧光测定CLC-3蛋白的表达部位。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。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 显微分离后弹力膜及内皮细胞层联合延迟消化培养法，体外培养牛角膜内皮细胞成功率高。采用DMEM/F12（1:1）培养基，细胞贴壁、生长、形态和活性最好。细胞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呈剂量相关性的促进培养的牛角膜内皮细胞增殖和移行, 联合应用重组人表皮生长因子（rhEGF）、碱性成纤维细胞生长因子（bFGF）对细胞增殖有协同作用。ET-1剂量依赖性增加CLC-3 mRNA及蛋白的表达，氯通道阻断剂 NPPB和CLC-3反义寡核苷酸浓度依赖性抑制ET-1促进细胞增殖、CLC-3 mRNA及蛋白表达的作用，并使细胞周期停滞于G1期，阻止细胞进入增殖期。
结论 ET-1通过开放氯通道，增加CLC-3 mRNA和蛋白的表达，促进培养的牛角膜内皮细胞增殖和移行。它和rhEGF、bFGF联合应用时有协同作用。CLC-3 AS-ODN和NPPB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。ET-1作为一种生长因子，有望成为治疗角膜内皮细胞损伤的新型药物之一。
The role of chloride channel-3 in the 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 and migration of cells and the role of chloride channel- 3 (CLC-3) in the process. Methods BCECs were cultured in vitro following two kinds of methods and with four kinds of culture medium. Injury model was used to observe the effect of ET-1 on cell migration. Immunohischemistry stainning, MTT assay and flow cytometry (FACS) were used to detect the expression of proliferating cell nuclear antigen (PCNA), cell proliferation, and cell cycle phase change. Immunofluorescence technique was used to localize the expression of CLC-3. The expression of CLC-3 mRNA and CLC-3 protein were tested by reverse transcription-polymerase chain reaction (RT-PCR) and western blot methods. Antisense oligodeoxynucleotide technique was used to observe the role of CLC-3 in the effect of ET-1 on BCECs. Results In the culture of BCECs in vitro, the methods of separating posterior elastic layer and endothelium of cornea under microscope, combined with delay digestion proved to succeed easily. DMEM/F12（1:1）culture medium should be adopted for best cell attachment, growth, morphologic changes and cell activity. The expression of endogeneous CLC-3 gene could be detected at the level of transcription and translation. CLC-3 protein expression localized in cell membrane and part of cell plasma cultured in vitro. ET-1 at the concentration from 10 pmol/L to 200 pmol/L promoted cell proliferation of cells in vitro and presented dosage correlation. The synergistic action on cell proliferation was presented if ET-1 was used together with rhEGF or bFGF. The expression levels of CLC-3 mRNA and CLC-3 protein increased with ET-1 in a dose dependent manner. Chloride channel inhibitor 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and antisense oligodeoxynucleotide of CLC-3 (CLC-3 AS-ODN) had inhibition effect on ET-1–induced cell proliferation and expression of CLC-3 mRNA and protein 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 CLC-3 protein, and promots the proliferation and migration of BCECs in vitro. There is the synergistic action on cell proliferation if ET-1 is used together with rhEGF or bFGF. By inhibiting CLC-3 chloride channel, NPPB and CLC-3 AS-ODN have the inhibition effect on ET-1–induced cell proliferation and result in that cell cycle is stagnant at G1 stage. Acting as a growth factor, ET-1 can be a new drug for corneal endothelium cells injury in future.
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