一种快速可靠筛选作用于细胞缝隙连接药物的方法建立

摘要

Aim To establish a fast and efficient drug screening method for gap junction ( GJ ) , which will provide a specific tool drug for GJ research and lay a good foundation to develop new drugs targeting GJ for the treatment of multiple diseases. Methods The pBI plasmid under the control of a bidirectional expressing Cx26/Cx32 was constructed. Using HeLa cells transfected with this Cx26/Cx32 cDNA. a HeLa cell model stably expressing Cx26/Cx32 was established, which could further form heteromeric GJ channels. Based on the c:ell model, the transfer of the fluorescent tracer between adjacent cells could be detected by dye transfer assay, confirming the functional gap junctional intrecellular communication ( GJIC ). In order to test whether the cell model could he used accurately to detect the alteration of GJ function by drug candidates, the effects of GJ activator retinoid acid ( RA ) and inhibitor oleamide on the function of GJ were also observed. Results The results of Western blot and dye transfer assay showed that the HeLa cells transfected with pBI plasmid containing Cx26/Cx32 cDNA. could express sufficient Cx26/Cx32 proteins and form heteromeric GJ function. Using this model, the alteration of GJ function by GJ activator and inhibitor was ohserved respectively. Conclusions With HeLa cells transfected with and stably expressing Cx26/Cx32. we demonstrate a means that can detect the effects of drugs on CJ function fast and efficiently by dye transfer assay. The establishment of this method will provide a useful technology for screening of drugs acting on GJ.%目的 建立一种可以快速有效地筛选能影响细胞缝隙连接(gap junction,GJ)功能药物的方法;为研制以GJ为靶点的新型药物提供技术手段.方法 构建能同时双向表达两种不同连接蛋白(connexins,Cxs)的质粒;用能表达Cx26/Cx32的质粒转染HeLa细胞,建立可以稳定表达Cx26/Cx32并形成异质性GJ的HeLa细胞模型.用荧光传递示踪法测定荧光示踪剂在细胞之间的传递,以此反映GJ的功能.观察公认的GJ激活剂--维甲酸(retinoid acid,RA)和GJ抑制剂--油酸酰胺(oleamide)对GJ功能的影响,检验该方法是否能够准确地检测出GJ功能.结果 Western blot和荧光传递示踪法的结果表明,转染含有Cx26/Cx32 cDNA质粒的HeLa细胞,可以稳定表达Cx26/Cx32;并能够形成可以传递荧光示踪剂在相邻细胞间的GJ.RA和油酸酰胺能分别增强和抑制荧光示踪剂的传递.结论 在转染Cx26/Cx32质粒并稳定表达Cxs的HeLa细胞上,用荧光传递示踪法测定GJ功能,可以快速准确地检测出药物对GJ的作用.该方法的建立为筛选作用于GJ的药物提供了一种有用的技术手段.