肝组织HBV总DNA和cccDNA阴性参考值的探讨

目的探讨肝组织乙型肝炎病毒（hepatitis B virus,HBV）总DNA（total DNA,tDNA）和共价闭合环状DNA（covalently closed circular DNA,cccDNA）的阴性参考值,为进一步研究实时定量PCR检测肝组织HBV tDNA和cccDNA的应用价值奠定基础。方法分别有88例HBsAg阳性/抗-HBc阳性、20例HBsAg阴性/抗-HBc阳性和19例HBsAg阴性/抗-HBc阴性慢性肝炎患者入选本研究。肝组织β-globin DNA、HBV tDNA和cccDNA采用实时荧光定量PCR检测,单个肝细胞HBV tDNA和cccDNA含量（copies/cell）=HBV tDNA实测值（copies）/β-globin DNA实测值（copies）和HBV cccDNA实测值（copies）/β-globin DNA实测值（copies）。肝组织HBVtDNA和cccDNA含量的计算单位定义为log_（10）copies/10~6cell。统计分析采用SPSS 13.0软件。结果肝组织HBVtDNA和cccDNA鉴别HBsAg阳性/抗-HBc阳性与HBsAg阴性/抗-HBc阴性慢性肝炎的ROC曲线下面积分别为0.909和0.891（P=0.000和0.000）,最佳截断值分别为5.772和4.883 log_（10） copies/10~6cells;鉴别HBsAg阳性/抗-HBc阳性与HBsAg阴性/抗-HBc阳性慢性肝炎的ROC曲线下面积分别为0.893和0.857（P=0.000和0.000.）,最佳截断值分别为5.559和4.630 log_（10） copies/10~6cells。就HBsAg阳性/抗-HBc阳性慢性肝炎而言,当肝组织HBV tDNA和cccDNA≥5.699和cccDNA≥4.699 log_（10） copies/10~6cells时,肝组织HBV tDNA与cccDNA含量之间均呈显著正相关（P=0.000和0.000）;当肝组织HBV tDNA〈5.699和cccDNA〈4.699 log_（10） copies/10~6cells时,肝组织HBV tDNA与cccDNA含量之间均无显著相关性（P=0.065和0.880）。结论肝组织HBV tDNA〈5.699和cccDNA〈4.699 log_（10） copies/10~6cells可能是合适的阴性参考值。

The negative reference cut-offs of intrahepatic hepatitis B virus total DNA and covalently closed circular DNA

Objective To determine the negative reference cut-offs for intrahepatic hepatitis B virus （HBV） total DNA（tDNA） and covalently closed circular DNA（cccDNA）.Methods Eighty eight patients with HBsAg-positive and anti-HBc-positive,20 patients with HBsAg-negative and anti-HBc-positive and 19 patients with HBsAg-negative and anti-HBc-negative chronic hepatitis were enrolled in the present study. Theβ-globin DNA,HBV tDNA and cccDNA in liver tissue were determined by real-time PCR.The content of single hepatocyte HBV tDNA（copies/cell）=HBV tDNA measured value（copies）/B-globin DNA measured value（copies）,and the content of single hepatocyte HBV cccDNA（copies/cell）=HBV cccDNA measured value（copies）/β-globin DNA measured value（copies）.The calculating unit of the content of intrahepatic HBV tDNA and cccDNA was log₁₀ copies/10⁶ cells.SPSS 13.0 software was used for statistical analyses.Results The areas under the curves of ROC of intrahepatic HBV tDNA and cccDNA for distinguishing HBsAg-positive and anti-HBc-positive chronic hepatitis from HBsAg-negative and anti-HBc -negative chronic hepatitis were 0.909 and 0.891（P=0.000 and 0.000）,respectively;and the optimal cut-offs were 5.772 and 4.883 log₁₀ copies/10⁶ cells,respectively.The areas under the curves of ROC of intrahepatic HBV tDNA and cccDNA for distinguishing HBsAg-positive and anti-HBc-positive chronic hepatitis from HBsAg-negative and anti-HBc-positive chronic hepatitis were 0.893 and 0.857（P=0.000 and 0.000）,respectively;and the optimal cut-offs were 5.559 and 4.630 log₁₀ copies/10⁶ cells,respectively.In HBsAg-positive and anti-HBc-positive chronic hepatitis,the content of intrahepatic HBV tDNA was positively correlated significantly with the content of intrahepatic HBV cccDNA when intrahepatic HBV tDNA≥5.699 and HBV cccDNA≥4.699 log₁₀ copies/10⁶ cells（P=0.000 and 0.000）;and the content of intrahepatic HBV tDNA was not correlated significantly with the content of intrahepatic HBV cccDNA when intrahepatic HBV tDNA<5.699 and HBV cccDNA<4.699 log₁₀ copies/10⁶ cells（P=0.065 and 0.880）. Conclusion Intrahepatic HBV tDNA<5.699 and HBV cccDNA<4.699 log₁₀ copies/10⁶ cells might be the rational negative reference cut-offs.