摘要
目的 :建立针对血清乙型肝炎病毒 (hepatitisBvirus,HBV)转录体的定量分析系统 .方法 :从血清中提取病毒DNA和RNA ,采用竞争性PCR技术、Southern blotting及分子克隆等技术 ,建立循环病毒DNA和RNA(转录体 )的定量分析系统 ,并应用其对抗病毒药物Lamivudine干预治疗后HBV感染患者血清中HBVDNA和RNA结构和数量的变化进行分析 .结果 :建立了两个针对X区RNA的定量分析系统 ,可检测带有全长型和顿挫型 3′端结构的转录体 .我们还建立了HBVX ,Core和X PreCore区段的DNA/RNA定量分析系统 ,三者分别与HBV基因复制的早、中、晚期对应 .此研究初步阐明了Lamivudine治疗期间患者血清中HBVDNA和转录体结构和数量的变化 .治疗 8wk以后 ,在Core和X PreCore区DNA拷贝数从 10 9·mL-1下降为 10 5·mL-1,下降幅度明显高于X区(下降至 10 7·mL-1) ,其比率更准确地反映了Lamivudine的作用效率 ,可用于疗效的评估 .转录体的拷贝数仅有小幅下降 ,采用锚定的Oligo(dT)引物检测的两种全长和顿挫型Ploy(A)RNA (10 5·mL-1)明显低于X区 (10 7·mL-1)RNA的拷贝数 ,提示在基因复制过程中前基因组RNA的Ploy(A)尾被去除 .结论 :这一分析系统可显示血清中HBV转录体含量及定量不同 3′端结构 。
AIM: To establish a quantitative system to characterize different RNA molecules (transcripts) of hepatitis B virus (HBV) in circulation. METHODS: Viral nucleic acids were extracted from serum samples and HBV DNA and RNA were characterized quantitatively by competitive PCR and RT-PCR procedures. RESULTS: A seroassay was established to characterize 3′-end structure of the full-length and truncated viral transcripts. Copy numbers of viral RNA/DNA segments corresponding to X, Core and X Precore regions were determined, representing early, middle and late stages of HBV gene replication, respectively. In addition, the data demonstrate dynamic changes in the circulating viral transcripts as well as DNA in their copy numbers and structures during lamivudine therapy. After a 8-week treatment, the copy numbers for C or PreC/X DNA segments decreased to 10 5·mL -1 from 10 9·mL -1 , significantly below that for X segment (from 10 9·mL -1 to 10 7·mL -1 ). There was no significant decrease in RNA copy numbers. Polyadenylated HBV RNA was also determined using anchored oligo (dT) primers targeting fRNA and trRNA . The copy numbers of fRNA and trRNA were 10 5 copies per mL of serum during most of the treatment period, significantly below that of X segment (10 7·mL -1 ). The excess of X segment RNA over fRNA levels suggested a packaging related removal of poly (A) 3′ ends. CONCLUSION: An assay for the detection of copy numbers and different 3′end structures of the circulating HBV transcripts is established, which provides a more precise approach to the detection of dynamic change of circulating HBV transcripts.
出处
《第四军医大学学报》
北大核心
2003年第8期673-677,共5页
Journal of the Fourth Military Medical University
基金
国家自然科学基金资助项目 (30 1 71 0 52 )
关键词
肝炎病毒
乙型
转录体
癌
肝细胞
血清
聚合酶链反应
拉米夫定
疗效评定
hepatitis B virus
transcript
carcinoma hepatocellular
serum
Polymerase Chain Reaction
lamivudine
therapy monitoring
