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为研究肝癌患者组织样本中HBV DNA核心启动子区(BCP区)和前C区(Pre C区)的基因突变多样性及其突变规律。收集第四军医大学附属西京医院2015年收治的192例HBV阳性的肝癌患者组织样本,PCR扩增HBV BCP/Pre C区DNA片段并进行测序分析,从测序失败的样本中随机抽取21例,采用构建单克隆文库后测序的方法进行分析。结果显示37.89%(72/190)的HBV阳性肝癌患者体内HBV病毒因呈现多种突变株混合感染的特点而导致PCR产物直接测序失败,经单克隆测序揭示,每一例失败样本的HBV DNA准种池中至少有2~11种突变株共同存在;突变株中缺失突变和插入突变的发生率高达80.95%;其它突变形式按照频率从高到低分别为A1762T/G1764A双突变90.48%,G1756C/T1803A/Δ(1 757~1 765)/Δ(1 824~1 832)四联突变80.95%,T1753C/A1762T/G1764A三联突变57.14%,A1762T/G1764A/G1896A三联突变42.86%,G1756C/Δ(1 757~1 765)双突变28.57%,T1753C/A1762T/G1764A/G1896A四联突变23.81%。由此可见,肝癌患者体内HBV病毒具有BCP/Pre C区DNA突变的多样性,这些缺失与插入突变是导致序列移码与PCR产物测序失败的直接原因。研究结果为HBV持续感染及基因突变检测、相关机制研究和个体化防治奠定了基础。
In order to study the gene mutation diversity and mutation of HBV DNA core promoter region (BCP region) and pre-C region (Pre C region) in tissue samples from patients with hepatocellular carcinoma (HCC). A total of 192 cases of HBV-positive hepatocellular carcinoma patients were collected from Xijing Hospital affiliated to the Fourth Military Medical University in 2015. The DNA fragments of HBV BCP / PreC region were amplified by PCR and sequenced. Twenty-one samples were randomly selected from the failed samples. Construction of a single library after sequencing method for analysis. The results showed that 37.89% (72/190) of HBV-positive patients with hepatitis B virus HBV infection due to the presence of a variety of mutant strains of mixed infection led to the direct sequencing of PCR products failed sequencing revealed by single nucleotide sequencing, each failed sample of HBV DNA quasispecies There were at least 2 ~ 11 kinds of mutants in the pool coexisting. The incidence of deletion mutation and insertion mutation in the mutant strain was as high as 80.95%. The other mutations were 90.48% of A1762T / G1764A double mutation and G1756C / T1803A The triple mutation of T1753C / A1762T / G1764A was 57.14%, and the triple mutation of A1762T / G1764A / G1896A was 42.86%, G1756C / Δ (1 757) ~ 1765) double mutation 28.57%, T1753C / A1762T / G1764A / G1896A triple mutation 23.81%. Thus, the HBV virus in patients with liver cancer has the diversity of DNA mutations in the BCP / Pre C region. These deletions and insertions are the direct causes of sequence mismatch and PCR product sequencing failure. The results of this study lay the foundation for the detection of persistent HBV infection and gene mutation, related mechanisms and individualized prevention and treatment.