人巨细胞病毒感染树鼩原代肝细胞的研究  

Human cytomegalovirus infecting primary Tupaia hepatocytes

作　　者：林海龙[1] 黄乐听[2] 陈洁[1] 夏蝉[1] 卢朝升[1] 

机构地区：[1]温州医科大学附属第二医院儿童感染科,浙江温州325003 [2]温州医科大学附属第二医院血液内科,浙江温州325003

出　　处：《中华医院感染学杂志》2016年第12期2669-2672,共4页Chinese Journal of Nosocomiology

基　　金：浙江省自然科学基金资助项目(LY13H150005);浙江省医药卫生科技基金资助项目(2013RCB011);温州市科技计划基金资助项目(Y20140055)

摘　　要：目的研究人巨细胞病毒AD169标准株(HCMV AD169)是否能感染树鼩肝原代细胞,为进一步研究人巨细胞病毒感染树鼩的动物模型研究打下基础。方法 2014年6月起取一只树鼩肝脏,分离并培养树鼩原代肝细胞,利用感染剂量MOI分别为0、0.1和1的HCMV AD169感染肝细胞,在不同时间点(12、24、48、72、120h)检测细胞培养液上清中病毒TCID50滴度,间接免疫荧光法检测肝细胞内HCMV AD169PP65蛋白表达,RT-PCR法检测培养肝细胞内HCMV AD169UL49mRNA,并对PCR结果进行测序并碱基序列比较。结果细胞培养液上清病毒TCID50滴度显示,HCMV AD169能在树鼩肝细胞内复制,间接免疫荧光法可以观察到肝细胞内病毒复制旺盛,RT-PCR和测序分析确证树鼩肝细胞内病毒为HCMV AD169。结论 HCMV AD169能感染体外培养的树鼩原代肝细胞,试实验具有一定的局限性,不能完全说明HCMV AD169感染树鼩动物的成功。OBJECTIVE To investigate whether HCMV AD169 can infect primary Tupaia hepatocytes,so as to provide reference for analyzing animal model of HCMV infecting primary Tupaia hepatocytes.METHODS In Jun.2014,the liver of a Tupaia was separated and primary Tupaia hepatocytes were cultured.HCMV AD169 with different multiplicity of infection（MOI）,including 0,0.1and 1,were used to infect hepatocytes.At different time points（12,24,48,72,120hours）,50 %tissue culture infection dose（TCID50）of cell culture was detected;indirect immunofluorescence was used to detect protein expression of HCMV AD169PP65 in hepatocytes;RT-PCR method was carried out to detect HCMV AD169 UL49 mRNA in hepatocytes,PCR results were sequenced and compared with base sequence.RESULTS According to TCID50,HCMV AD169 can duplicate in Tupaia hepatocytes.Indirect immunofluorescence showed active duplication of virus in hepatocytes.RT-PCR and sequencing analysis confirmed the virus in Tupaia hepatocytes was HCMV AD169.CONCLUSION HCMV AD169 can infect vitro-cultured Tupaia hepatocytes.But this experiment is limited to some extent,that it can not fully prove the success of HCMV AD169 infecting Tupaia hepatocytes.

关 键 词：树鼩 原代肝细胞 人巨细胞病毒 测序 

分 类 号：R373.2[医药卫生—病原生物学]