利用人支气管上皮细胞系分离和培养人博卡病毒  

Isolation and cell culture of Human bocavirus（HBoV）by Human bronchial epithelial cell lines

作　　者：林峰[1] 滕灵方[1] 郑美云[1] 郑昌华[1] 吴锋[1] 李桦[1] 郑敏巧[1] 曾爱平[1] 黄恩佩[1] 莫一涵[1] 侯建毅[1] 

机构地区：[1]温州医学院附属温岭医院,浙江温州317500

出　　处：《中华实验和临床病毒学杂志》2009年第6期437-439,共3页Chinese Journal of Experimental and Clinical Virology

基　　金：国家自然科学基金（No.30771913）;国家高技术研究发展“八六三”计划基金（No.2007AA02Z400）.

摘　　要：目的研究人博卡病毒（HBoV）在呼吸道感染中致病性及机理。方法利用人支气管上皮细胞系进行HBoV分离及培养，通过电镜对培养细胞中HBoV的形态及感染细胞进行研究，并采用逆转录PCR（RT-PCR）方法，检测HBoV在细胞中转录产物mRNA，同时对扩增产物进行测序及分析。结果将含有HBoV阳性痰液标本无菌接种人支气管上皮细胞系，出现2例致使细胞发生明显的病变（CPE）。电镜观察在细胞质中散在六角形或圆形的病毒粒子，大小18-26nm之间，大部分是20nm。RT-PCR扩增产物经电泳检测与预期结果相符是295bp，扩增产物测序结果与GenBank中HBoV序列比较，核苷酸同缘性99％，氨基酸同缘性100％。结论HBoV感染人支气管上皮细胞后能够在该细胞内增殖，并产生特征性生物学改变。本研究为今后HBoV致病性及免疫应答谱研究奠定基础。Objective To investigate pave a way for studying pathogenicty of HBoV. Methods Isolation and cell culture of HBoV by human bronchial epithelial cell line, which was founded in our laboratory. The morphology of the virus were primarily studied with a transmission electron microscope. In addition, transcript mRNA was detected in human bronchial epithelial cells, which was passaged and infected within HBoV, using the reverse-transcription polymerase chain reaction（RT-PCR）. The amplified products nucleotide sequence of HBoV were sequencing and sequence analysis. Results Cytopathic effect （CPE） was observed after the aseptic residue of filtration of 2 case sputum specimens with HBoV, which was inoculated to the human bronchial epithelial cell line. The virus particles were observed in the cytoplasm, which were hexagonal or spherical in shape and 18-26 nm in diameter, bulk was 20 nm. cDNA amplicon obtained 295 bp fragment results of electrophoresis bands as same as NS1 region of the conserved matrix gene of publish sequence of HboV. PCR products nucleotide sequence of HboV were compared withcorresponding HboV GeneBank sequences. The comparison/alignment and construction of phylogenetic trees also point to an affiliation of the parvovirus to the species HBoV. Conclusion Isolation and identification of HBoV could be done in the human bronchial epithelial cell, and we found some characterizing CPE in the human bronchial epithelial cell after HBoV infection. The above studies pave a way for studying pathogenicty of human bocavirus.

关 键 词：细小病毒科 支气管 上皮细胞 细胞 培养的 

分 类 号：R5[医药卫生—内科学]