基于pH值敏感的荧光染料分析腺病毒诱导T淋巴细胞胞内体膜的裂解  

A novel assay for analysis of adenovirus-mediated endosome lysis of T lymphocytes

作　　者：张文峰[1] 李壮华[2] 陈镜塘[2] 邵红伟[1] 陈业诚 黄树林[1] 

机构地区：[1]广东药科大学生命科学与生物制药学院,广东省生物技术候选药物研究重点实验室,广东广州510006 [2]东莞市人民医院,广东东莞523059

出　　处：《微生物学报》2016年第11期1776-1785,共10页Acta Microbiologica Sinica

基　　金：国家自然科学基金(31400149,31300737);广东省科技计划项目(2014A020212462);广东省自然科学基金(2014A030313586)~~

摘　　要：【目的】探索基于pH值敏感的荧光染料分析腺病毒裂解T淋巴细胞胞内体膜的实验方法。【方法】本文以Jurkat细胞(T淋巴瘤细胞)为靶细胞,将pH值敏感的荧光染料pHrodo dextran与5型腺病毒(Ad5)共同孵育Jurkat细胞,对pHrodo dextran孵育的浓度与时间进行了优化,利用激光共聚焦显微镜分析胞内相对平均荧光强度百分比随时间的变化情况,反映Ad5诱导胞内体膜裂解情况。【结果】研究结果表明,在pHrodo dextran终浓度为80μg/m L,孵育时间为10 min条件下,在病毒感染后的30 min,相对平均荧光强度百分比出现显著下降;利用巴佛洛酶素A1抑制胞内体膜质子泵活性后,相对平均荧光强度百分比出现轻微下降。【结论】建立了基于pHrodo dextran分析腺病毒诱导T细胞胞内体膜裂解的新方法。[Objective] To analyze adenovirus-mediated endosome lysis of T cells, we developed a novel approach based on pHrodo dextran（pH-sensitive fluorescent dye）. [Methods] After incubating Jurkat cells（T cell leukemia）with serotype 5 adenovirus（Ad5） and pHrodo dextran, we determined the optimal incubation time and concentration of pHrodo dextran. To assess viral lysis of the endosome, we monitored the ratio changes of mean fluorescence intensity in different time points by laser scanning confocal microscopy. [Results] After incubating Jurkat cells with Ad5 and 80 μg/m L pHrodo dextran for 10 minutes, we observed the fluorescence intensity was significantly reduced at 30 minutes compared with that of endosomes at 0 minute. However, we found the mean fluorescence intensity was only slightly reduced by inhibiting V-ATPase with the bafilomycin A1 treatment. [Conclusion] The method based on pH-sensitive dye can be used to analyze the adenovirus-mediated endosome lysis of T cells.

关 键 词：腺病毒 胞内体 胞内运输 T淋巴细胞 

分 类 号：R392[医药卫生—免疫学]