HBV感染患者外周血单个核细胞HBV DNA和cccDNA的检测  

Detection of ccc DNA and HBV DNA in peripheral blood mononuclear cells of patients with HBV infection by in situ hybridization

作　　者：刘畅 钟彦伟[2] 徐元勇 邸凤林 吴贻琛[2] 刘鸿博 李浩 姜棋予 王志杰[2] 

机构地区：[1]解放军疾病预防控制所全军疾病监测中心,北京100071 [2]解放军302医院临床研究管理中心,北京100039 [3]河北北方学院检验学院寄生虫教研室,河北张家口075000

出　　处：《中华疾病控制杂志》2016年第9期888-892,共5页Chinese Journal of Disease Control & Prevention

基　　金：北京市自然科学基金(7122126)

摘　　要：目的探讨乙型肝炎患者外周血单个核细胞(peripheral blood mononuclear cells,PBMC)能否感染乙型肝炎病毒(hepatitis B virus,HBV)并进行复制。方法选取137例住院患者作为研究对象,采集血液标本,分离提取外周血单个核细胞,使用细胞甩片法固定细胞,采用实时荧光定量聚合酶链式反应技术(real-time quantitative polymerase chain reaction,QPCR)和滚环扩增结合原位跨缺口原位聚合酶链式反应技术检测HBV共价闭合环状DNA(covalently closed circular DNA,ccc DNA);免疫组织化学染色法检测HBV DNA和HBV ccc DNA表达。结果 137例标本中,68.6%(94/137)的标本经免疫组化染色后检测出HBV DNA的表达;83.9%(115/137)的标本原位杂交和滚环扩增PCR后检出HBV ccc DNA阳性信号;血清HBV DNA浓度高于106 copies/ml的患者PBMC中HBV ccc DNA阳性率为95.1%,低于106 copies/ml的阳性率为51.4%。结论高浓度外周血HBV DNA是HBV感染外周血单个核细胞的危险因素,通过原位聚合酶链式反应方法能够更灵敏地检测HBV DNA和ccc DNA在PBMC中的表达,为进一步研究证实PBMC感染导致HBV母婴传播途径的分子机制奠定基础。Objective To investigate whether hepatitis B virus （HBV） can infect and copy in the peripheral blood mononuclear cell （PBMC） in patients with chronic hepatitis B. Methods 137 inpatient attenders＇ peripheral blood was selected;mononuclear cells were isolated from peripheral blood;cells were fixed using cell rejection slice method; HBV DNA and cccDNA were detected with Real-time Quantitative polymerouse chain rection（QPCR） and rolling circle amplifi- cation combined with in situ cross gap PCR immunohistochemical staining method was used to detect HBV DNA and HBV cccDNA expression in PBMC. Results In 137 cases of specimen, after immunohistochemical staining, 68.6% （94/137） cases were detected the expression of HBV DNA, 83.9% （115/137） cases were deteced of HBV cccDNA positive signal by in situ hybridization and rolling circle amplification PCR; the positive rate of HBV cccDNA in PBMC of patients with se- rum HBV DNA concentration with higher than 106 copies/ml was 95. 1%, the positive rate of less than 106 copies/ml was 51.4%. Conclusions HBV can infect the peripheral blood mononuclear cells and replicate in it, this study lays the foun- dation for further study of the PBMC infection leading to intrauterine infection of HBV.

关 键 词：肝炎病毒 乙型 DNA 原位标记 

分 类 号：R373.21[医药卫生—病原生物学] R181.1[医药卫生—基础医学]