摘要
目的建立评价副溶血弧菌毒力的Raw-264细胞模型,并以此评价副溶血弧菌临床分离株RIMD2210633株与其重要致病相关基因opaR基因敲除株的毒力。方法以Raw-264细胞为靶细胞,RIMD2210633株及其opaR基因敲除株为效应细胞,通过优化条件,利用CytoTox 96Non-Radioactive Cytotoxicity Assay建立细胞模型。结果以5倍于靶细胞的菌量感染靶细胞,37℃孵育1.5 h,离心所得上清稀释10倍后,加底物液避光显色10 min,反应条件最佳。opaR基因敲除后,菌株的Raw-264细胞毒性升高,并有统计学差异。结论利用副溶血弧菌标准强毒株RIMD2210633菌株建立了评价副溶血弧菌毒力的RAW-264细胞模型,并利用此平台比较了该毒株与其opaR基因敲除株的毒力,为研究副溶血弧菌的致病机制奠定了基础。
Objective To establish a Raw -264 cell model for evaluating virulence of Vibrio parahaemolyticus. Methods The CytoTox 96 Non - Radioactive Cytotoxicity Assay Kit was employed to test the Vibrio parahaemolytieus - induced cytotoxicity against Raw264 cells. Results The protocols were optimized as follows： Vibrio parahaemolyticus cells and Raw264 cells were incubated for 1.5 h at 37℃ with a MOI of 5 ; the mixture was then pelleted, and the 10 - fold diluted superuatant was incubated with the substrate for 10 min. The cytotoxicity against Raw- 264 cells determined for the opaR mutant was significantly higher than that for the wild - type strain. Conclusions A Raw - 264 cellular model was established for evaluating virulence of Vibrio t）arahaemolvticus.
出处
《武警医学》
CAS
2013年第9期796-799,共4页
Medical Journal of the Chinese People's Armed Police Force
基金
国家自然科学基金(31170127)
国家重点基础研究发展计划"973"项目(2009CB522604)
