钠钙交换体基因稳定转染及药物筛选细胞模型的建立VIP专享VIP免费

史上最快最全的网络文档批量下载批量上传，尽在：http://item.taobao.com/item.htm?id=9176907081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。关键词：NCX1.1；NCX1.4；NCX1.5；NCX 抑制剂 KB-R7943；膜片钳Establishment and Evalution of Cell Model Targeted at Sodium-Calcium ExchangerLong 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-pc...