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目的阐明乙型肝炎病毒(hepatitis B virus,HBV)相关肝细胞癌的癌组织与癌旁组织中HBV cccDNA全基因组的点突变模式及其与HBV cccDNA和总DNA含量的关系,寻找可以作为肝细胞癌(hepatocellular carcinoma,HCC)患者预后判断指标的突变位点。方法取23例HCC患者术后肝癌与癌旁组织各1份,采用Qiagen法提取DNA,采用滚环扩增HBV cccDNA,采用Taqman探针法定量测定HBV cccDNA和总DNA。采用PCR分段扩增HBV cccDNA全基因组,PCR产物纯化后测序,确定其基因型,并与标准野生株序列比对,确定HBV cccDNA的突变位点。结果肝癌与癌旁组织中HBV cccDNA突变频率较高的位点集中于X和C区,肝癌与癌旁组织突变个数之和大于10的位点有26个。G1719突变组HBV cccDNA和总DNA的含量均低于T1719野生型组(t=2.449,P<0.05和t=2.525,P<0.05),G1719突变组患者术后生存时间短于T1719野生型组(χ2=8.56,P<0.05),并且T1719G突变与患者BCLC分级相关,G1719突变组更倾向于较高的BCLC分级(χ2=6.296,P<0.05)。结论 T1719G突变可能成为HCC患者术后预后的早期预测因素,值得进一步研究。
Objective To elucidate the point mutation pattern of HBV cccDNA genome and its relationship with HBV cccDNA and total DNA content in cancer tissues and adjacent tissues of hepatitis B virus (HBV) -related hepatocellular carcinoma (HCC) The prognosis of hepatocellular carcinoma (HCC) prognostic indicators of the mutation sites. Methods Twenty-three patients with HCC were divided into two groups, one each with hepatocellular carcinoma and adjacent non-cancerous tissues. Qiagen method was used to extract DNA. HBV cccDNA was amplified by rolling circle and HBV cccDNA and total DNA were quantitatively determined by Taqman probe. The entire genome of HBV cccDNA was amplified by PCR. The PCR product was purified and sequenced to determine its genotype. The results were compared with the standard wild-type sequence to determine the mutation site of HBV cccDNA. Results The frequency of HBV cccDNA mutation in HCC and adjacent tissues was concentrated in X and C regions. There were 26 sites with more than 10 mutations in HCC tissues and paracancerous tissues. The levels of HBV cccDNA and total DNA in G1719 mutation group were lower than those in T1719 wild type group (t = 2.449, P <0.05 and t = 2.525, P <0.05). The survival time of G1719 mutation group was shorter than that of T1719 wild type group χ2 = 8.56, P <0.05), and the mutation of T1719G was associated with the BCLC grade of patients. The G1719 mutation group was more likely to have higher BCLC grade (χ2 = 6.296, P <0.05). Conclusion The T1719G mutation may be an early prognostic factor for postoperative prognosis of HCC patients and warrants further study.