摘要
目的探讨慢性乙型肝炎患者肝组织HBV共价闭合环状DNA(cccDNA)、肝组织总HBV DNA(HBV tDNA)与血清HBV DNA之间的相关性及其与临床的关系。方法78例慢性乙型肝炎患者入选本研究。肝组织β—globin DNA、HBV cccDNA和HBV tDNA采用实时荧光定量PCR方法检测,平均每个肝细胞HBV cccDNA和HBV tDNA含量(拷贝/cell)=HBV cccDNA(实测值)/β-globin DNA(实测值)和HBV tDNA(实测值)/β-globin DNA(实测值),肝组织HBV cccDNA和HBV tDNA含量的计算单位定义为log10拷贝/10^6 cell;采用实时荧光定量PCR、ELISA法检测血清HBV DNA和HBV标志物;采用免疫组织化学方法检测肝细胞中HBsAg和HBcAg的表达。统计分析采用pearson相关分析及t检验。结果(1)肝组织HBV cccDNA与HBV tDNA定量呈正相关(r=0.696,P〈0.001);肝组织HBV cccDNA与血清HBV DNA定量呈正相关(r=0.304,P%0.01);肝组织HBV tDNA与血清HBV DNA定量呈正相关(r=0.341,P〈0.01);(2)肝细胞内HBcAg定性检测阳性患者的血清HBV DNA定量明显高于阴性患者,且差异有统计学意义(P〈0.05);肝细胞内HBcAg定性检测阳性患者与阴性患者的肝组织HBV cccDNA和HBV tDNA定量差异均无统计学意义和(P均〉0.05);(3)肝细胞内HBsAg定性检测阳性患者的血清HBV DNA定量明显高于阴性患者,且差异有统计学意义(P〈0.05);肝细胞内HBsAg定性检测阳性患者与阴性患者的肝组织HBV cccDNA和HBV tDNA定量差异均无统计学意义(P〉0.05);(4)HBeAg(+)/抗HBe(-)患者血清HBV DNA定量明显高于HBeAg(-)/抗-HBe(+)患者,且差异有统计学意义(P〈0.05);HBeAg(+)/抗-HBe(-)患者肝组织HBV cccDNA和HBV tDNA定量与HBeAg(-)/抗-HBe(+)患者比较差异均无统计学意义(均P〉0.05);(5)肝组织HBV cccDNA、HBV tDNA以及血清HBV DNA三者与肝脏炎症活动度及纤维化程度均无显著相关性(P〉0.05)。结论(1)血清HBV DNA定量结果并不一定能完全反映患者肝组织中HBV cccDNA和HBV tDNA含量,尤其在血清HBV DNA%500拷贝/mL时,肝组织中仍存在HBV cccDNA和HBV tDNA,且含量大小不等。(2)肝细胞内HBcAg定性检测阳性或者HBsAg定性检测阳性患者的血清HBV DNA定量均明显高于阴性患者;而两者的肝组织HBV cccDNA和HBV tDNA定量均没有显著差异;(3)HBeAg(+)/抗-HBe(-)患者血清HBV DNA定量明显高于HBeAg(-)/抗-HBe(+)患者,而两者的肝组织HBV cccDNA和HBV tDNA均没有显著差异;(4)肝组织HBV cccDNA、HBV tDNA及血清HBV DNA与肝脏炎症活动度和纤维化程度均无显著相关性。
Objective To investigate the relationship among covalent closed circular DNA (cccDNA), HBV total DNA (tDNA) and serum HBV DNA and their clinic-pathological significances in patients with chronic hepatitis B. Methods Seventy eight patients with chronic hepatitis B were enrolled in this study. The β- globin DNA, HBV cccDNA and HBV tDNA in liver tissue were detected by real-time PCR. The content of single hepatocyte HBV cccDNA and HBV tDNA (cop- ies/cell) equals to HBV cccDNA (measured value)/β-globin DNA measured value(copies), and HBV tDNA measured value (copies)/β-globin DNA measured value(copies). The content of intrahepatic HBV cccDNA and HBV tDNA was recorded as log10 copies/10^6 cell. Real time PCR and ELISA were performed to determine serum HBV DNA and HBV markers respec- tively. The expression of HBsAg and HBcAg in hepatocytes was detected by immunohistochemical method. SPSS 13.0 soft ware was adopted for statistical analysis. Results (1) There was a positive correlation between the hepatocyte HBV cccDNA and HBV tDNA (r = 0. 696, P〈(0. 001 ) , Similar positive correlation was found between hepatocyte HBV cccDNA and serum HBV DNA (r= 0. 304, P〈0.01 ) ,. There was a positive correlation between hepatocyte HBV tDNA and serum HBV DNA (r= 0. 341, P〈0. 01) as well. (2) Serum HBV DNA in patients with intrahepatic HBcAg( + ) was higher than that in patients with intrahepatic HBcAg( - ), and there was a significant difference(P〈0.05). There was no significant differ enee between hepatocyte HBV cccDNA and HBV tDNA in patients with intrahepatic HBcAg( + ) and intrahepatic HBcAg ( - )with P〉0.05 respectively. (3) Serum HBV DNA with intrahepatic HBsAg( + ) was higher than that in patients with intrahepatic HBsAg( - ), and there was a significant difference(P〈0.05). There was a positive correlation between hepa tocyte HBV cccDNA and HBV tDNA in patients with intrahepatic HBsAg( + ) and intrahepatic HBsAg( - ) with P〉0.05 respectively. (4) Serum HBV DNA in patients with HBeAg ( + )/ anti-HBe ( - ) was higher than that in patients with HBeAg( - )/anti-HBe( + ), and there was a significant difference(P〈0. 05). There was no significant difference between hepatocyte HBV cccDNA and HBV tDNA in patients with HBeAg ( + )/anti-HBe ( - ) and HBeAg ( - )/anti-HBe ( + ) with P〉0.05 respectively. (5) There was no significant correlation between hepatocyte HBV cccDNA, HBV tDNA and se- rum HBV DNA and the levels of liver inflammation activity and fibrosis (P〉0.05). Conclusion (1) Serum HBV DNA cannot fully reflect the content of hepatocyte HBV cccDNA and HBV tDNA. Even when serum HBV DNA level was lower than 500 copies/mL, hepatocyte HBV cccDNA could still be detected with the maximum difference of 10 times of the con- tents. (2) Serum HBV DNA in patients with intrahepatic HBcAg( + ) or HBsAg( + ) was both higher than that in patients with intrahepatic HBcAg ( - ) or HBsAg ( - ), and there was no significant difference between their hepatocyte HBV cccDNA and HBV tDNA . (3) Serum HBV DNA in patients with HBeAg ( + )/anti- HBe( - ) was higher than that in patients with HBeAg ( - )/anti HBe ( + ), and there was no significant difference between hepatocyte HBV cccDNA and HBV tDNA. (4) There was no significant correlation between hepatocyte HBV cccDNA, HBV tDNA and serum HBV DNA and the levels of liver inflammation activity and fibrosis.
出处
《肝脏》
2012年第6期381-384,共4页
Chinese Hepatology