钙激活氯离子通道在气道杯状细胞合成黏蛋白中的促进作用_医学论文.docVIP

钙激活氯离子通道在气道杯状细胞合成黏蛋白中的促进作用_医学论文 钙激活氯离子通道在气道杯状细胞合成黏蛋白中的促进作用_医学论文 作者：宋立强，戚好文，李妍，王吉村，林爱华 【关键词】 杯状细胞 　　【Abstract】 AIM: To investigate the effects of human calciumactivated chloride channel 1 (hCLCA1) on synthesis of mucin in airway goblet cells. METHODS: Recombined eukaryotic expression plasmid pIRES2EFGP/hCLCA1, which contained fluorescence tag and G418 screening flag was constructed. The plasmid was stably transfected into NCIH292 cell line, which was regarded as goblet cell model in vitro. Transfected NCIH292/hCLCA1 cells were identified by fluorescence microscopy and hCLCA1 mRNA was detected by RTPCR. Cells were divided into four groups with or without hCLCA1 inhibitor NFA: ① NCIH292 ② NCIH292/hCLCA1 ③ NCIH292+NFA ④ NCIH292/hCLCA1+NFA. The proliferation of the cells in all groups was detected using MTT colorimetry. The expression of mucin MUC5AC protein and mRNA was examined with PAS staining and RTPCR assay, respectively. RESULTS: NCIH292/hCLCA1 cells that stably expressed hCLCA1 were obtained. The proliferation capability, mucin protein expression and mRNA level of NCIH292/hCLCA1 cells were all significantly higher than those of untransfected cells. NFA markedly inhibited the above changes of NCIH292/hCLCA1 cells. CONCLUSION: hCLCA1 promotes the proliferation and mucin synthesis in airway goblet cells. This study indicates that the inhibition of hCLCA1 may be a potential treatment for asthma. 　　【Keywords】 asthma airway epithelial cells goblet cells chloride channel 　　【摘要】 目的： 探讨人激活Cl通道I型（hCLCA1）在气道杯状细胞合成黏蛋白中的调控作用. 方法： 以人气道黏液上皮细胞系NCIH292作为杯状细胞特性研究的体外模型，构建携带荧光蛋白标签的真核表达质粒pIRES2EFGP/hCLCA1，转染NCIH292细胞，用G418筛选稳定表达hCLCA1的细胞NCIH292/hCLCA1. 对被转染细胞采用荧光显微镜和RTPCR法检测hCLCA1 mRNA. 根据NFA（hCLCA1阻断剂）干预与否，将细胞分为4组： ① NCIH292；② NCIH292/hCLCA1；③ NCIH292+NFA；④ NCIH292/hCLCA1+NFA. MTT法检测各组细胞的增殖活性；PAS特染法检测各组细胞黏液糖蛋白的表达状况, RTPCR法检测黏蛋白MUC5AC mRNA水平. 结果： 经过500g/L的G418筛选，证实获得NCIH292/hCLCA1细胞. 对各组细胞检测结果表明，NCIH292/hCLCA1细胞的增殖活性、黏液糖蛋白表达量及MUC5AC mRNA水平均显著高于亲本细胞，并且NFA能有效抑制NCIH292/hCLCA1的这些生物活性