钠钙交换体基因稳定转染及药物筛选细胞模型的建立.doc

钠钙交换体基因稳定转染及药物筛选细胞模型的建立 龙雁，袁辉，王晓良* 中国医学科学院北京协和医学院 药物研究所 先农坛街1号，北京，100050 摘要：目的 构建三种不同亚型的CHO-NCX稳定转染细胞株，以便筛选可能的钠钙交换体（NCX）调节剂。 方法 采用脂质体法将NCX目的基因稳定导入CHO细胞中，检测稳定转染细胞株中NCX的蛋白表达和电流大小，并观察温度和NCX抑制剂KB-R7943对电流的影响。 结果 Western blot结果显示与CHO-pcDNA3.1空白细胞相比，CHO-NCX稳定转染细胞株中NCX蛋白的表达量显著升高；电生理结果提示CHO-NCX稳定转染细胞株中NCX电流远大于CHO-pcDNA3.1空白细胞；温度从22℃升高到35℃时，NCX电流显著增大；NCX抑制剂KB-R7943对INCX具有显著抑制作用。 结论 本试验成功的建立了特异性表达NCX1.1，NCX1.4，NCX1.5三种亚型的细胞模型，为脑缺血等疾病的研究提供了良好的药物筛选平台。 NCX1.1；NCX1.4；NCX1.5；NCXKB-R7943；膜片钳 Establishment and Evalution of Cell Model Targeted at Sodium-Calcium Exchanger Long Yan, Yuan Hui, Wang Xiaoliang (Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China) ABSTRAT: OBJECTIVE To develop potential NCX modulator with 3 different kinds of CHO-NCX cells which stably expressed NCX proteins. METHODS The NCX genes were ligated with pcDNA3.1+ vector and then stably introduced into CHO cells by liposome. Using western blot and patch clamp technique to measure the expression levels of NCX proteins and NCX currents, after which to observe the magnitude of NCX currents when temperature changed and NCX inhibitor utilized. RESULTS Compared with blank CHO-pcDNA3.1 cells, the protein amounts of stably transfected CHO-NCX cells were significantly increased; the currents of CHO-NCX cells were much higher than that of blank CHO-pcDNA3.1 cells. When temperature changed from 22℃ to 35℃, the currents of CHO-NCX cells up-regulated notably, after NCX inhibitor KB-R7943 added, the currents of CHO-NCX cells decreased significantly. CONCLUSION Our study successfully constructed a cell model which expressed a specific kind of NCX isoform in CHO cells, which provided a useful tool to search new drugs of cerebral ischemia targeted at NCX. KEY WORDS: NCX1.1; NCX1.4; NCX1.5; NCX inhibitor KB-R7943; patch clamp 作者简介：龙雁，女，博士研究生 Tel：(010 longyan@imm.ac.cn 钠钙交换体（NCX）是细胞膜上具有9个跨膜片段的一种双向转运体，根据细胞膜内外离子浓度梯度和膜电位的不同，NCX可以通过正向转运（Ca2+ 外流）或者反向转运（Ca2+