Effects of circ_0063865 silencing on the malignant phenotype of esophageal squamous cell carcinoma / 中华老年医学杂志

Objective:To investigate the expression of circ_0063865 in esophageal squamous cell carcinoma(ESCC)tissues and cells and its effect on the biological properties of the cells.Methods:The loop structure and stability of circ_0063865 were identified by Sanger sequencing, back-to-back primer validation and the ribonuclease R(Rnase R)tolerance assay.The expression of circ_0063865 was detected by RNA fluorescence in situ hybridization in an ESCC tissue microarray and its clinical relevance was analyzed.The expression levels of circ_0063865 in a normal esophageal epithelial cell line and ESCC cell lines were measured by real-time quantitative polymerase chain reaction(RT-qPCR). Cell counting Kit-8, the colony formation assay, the scratch assay, the transwell invasion assay and flow cytometry were used to detect the effects of circ_0063865 on cell proliferation, migration, invasion abilities and apoptosis, respectively.Results:The loop formation of circ_0063865 was verified by Sanger sequencing, back-to-back primer and Rnase R tolerance assays.The results of RNA fluorescence in situ hybridization showed that the mean fluorescence intensity of circ_0063865 expressed in ESCC tissues was significantly higher than in its paired paracancerous normal tissues( t=2.267, P<0.05). The expression of circ_0063865 was significantly associated with lymph node metastasis( χ2=4.356, P<0.05). The average overall survival time of patients with high circ_0063865 expression ESCC was lower than that of patients with low circ_0063865 expression ESCC.RT-qPCR results demonstrated that, compared with HEEC, circ_0063865 expression was elevated in ESCC cell lines( F=18.413, P<0.05). In addition, after circ_0063865 knockdown, the proliferation, migration and invasion abilities of KYSE-30 and KYSE-150 cells were significantly decreased, and the level of apoptosis was significantly increased(both P<0.05). Conclusions:The expression of circ_0063865 in ESCC is high, and changes in its expression are significantly correlated with lymph node metastasis.Additionally, circ_0063865 can promote the proliferation, migration and invasion of ESCC cells.

Functional cure of hepatitis B from the perspective of hepatitis B virus covalently closed circular DNA / 临床肝胆病杂志

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) is the template for HBV transcription and replication and exists stably in hepatocytes in the form of minichromosome. Based on the mechanism of cccDNA inactivation or clearance and the development of related drugs, this article introduces the current knowledge on the formation, transcription, and degradation of cccDNA and reviews the research advances in cccDNA-targeted drugs and biological techniques.

Establishment of a droplet digital PCR method for the detection of hepatitis B virus covalently closed circular DNA / 临床肝胆病杂志

ObjectiveTo establish a droplet digital PCR (ddPCR) method for detecting hepatitis B virus (HBV) covalently closed circular DNA (cccDNA). MethodsHBV cccDNA standard substance was constructed, and HBV cccDNA primers and probes were designed based on the structural differences between HBV cccDNA and relaxed circular DNA (rcDNA). HBV plasmid was amplified to obtain HBV cccDNA standard substance, and a ddPCR detection method was established with the standard substance after gradient dilution as the template for HBV cccDNA detection; the limit of detection and repeatability of this method were analyzed. Liver tissue samples were collected from 20 patients who attended Beijing YouAn Hospital, Capital Medical University, from June 2017 to October 2020, all of whom were diagnosed with HBV infection, and DNA of the samples was extracted and digested with plasmid-safe ATP-dependent DNA enzyme to obtain HBV cccDNA template; the ddPCR detection method was evaluated in clinical samples and was compared with the quantitative real-time PCR (qPCR) detection method. The chi-square test was used for comparison of categorical data between the two groups. ResultsThe HBV cccDNA detection method based on ddPCR was established, which accurately detected HBV cccDNA in standard substance after gradient dilution, with a limit of detection of 1 copy/μl, and the coefficients of variation of 1×103, 1×102, and 1×101 copies/μl standard substances were 441%, 3.98%, and 5.09%, respectively. HBV cccDNA was detected in the samples of 20 patients with HBV infection; the ddPCR detection method detected HBV cccDNA in 17 patients, with a positive rate of 85%, while the qPCR detection method detected HBV cccDNA in 11 patients, with a positive rate of 55%, and there was a significant difference between the two methods (χ2=4.286, P=0038). ConclusionThe established ddPCR method for detecting HBV cccDNA has a low limit of detection and good repeatability, which provides an effective tool for further clinical detection.

Effect of circRNA hsa_circ_0091579 on the proliferation, migration, and invasion of hepatoma cells / 临床肝胆病杂志

ObjectiveTo investigate the expression of circular RNA (cirRNA) hsa_circ_0091579 in human hepatocellular carcinoma (HCC) cell lines and its effect on the proliferation, migration, and invasion of HCC cells. MethodsHuman HCC cell lines SMMC-7721, Huh-7, MHCC-97H, and HepG2 and normal human liver cell line HL-7702 were cultured in vitro. RNA was extracted from cells and quantitative real-time PCR (qRT-PCR) was used to measure the expression of hsa_circ_0091579 in HCC cell lines and normal human liver cell line. Small interfering RNA (siRNA) was designed for the cyclic splicing site of hsa_circ_0091579, and HepG2 and Huh-7 cells were transfected with hsa_circ_0091579 siRNA in vitro; qRT-PCR was used to verify transfection efficiency. The experiment was divided into siRNA group (transfected with hsa_circ_0091579 siRNA) and NC group (transfected with negative control siRNA), and CCK-8 assay, flow cytometry, wound healing assay, and Transwell assay were used to investigate the effect of hsa_circ_0091579 on cell proliferation, apoptosis, migration, and invasion in HepG2 and Huh-7 cells. Dual luciferase reporter gene assay was used to verify the predicted target. The t-test was used for comparison of continuous data between the two groups. ResultsCompared with the normal human liver cell line HL-7702, the HCC cell lines SMMC-7721, Huh-7, MHCC-97H, and HepG2 had a significant increase in the expression level of hsa_circ_0091579 (t=14.27, 36.34, 26.70, and 36.16, all P＜0.001). Compared with the NC group, hsa_circ_0091579 siRNA effectively silenced hsa_circ_0091579 in HepG2 and Huh-7 cells (t=14.22 and 27.20, P=0.005 and 0.001). CCK-8 assay and flow cytometry showed that compared with the NC group, the siRNA group had a significant reduction in the proliferative activity of HepG2 and Huh-7 cells and a significant increase in apoptosis rate (all P＜0.05); wound healing assay and Transwell assay showed that compared with the NC group, the siRNA group had significant reductions in the migration and invasion abilities of HepG2 and Huh-7 cells (t=19.63, 13.61, 20.75, and 18.45, P=0.003, 0.005, 0.002, and 0.003). Luciferase reporter assay showed that compared with the NC group, miR-149, miR-490-5p, and miR-502-5p significantly reduced the activity of wild-type luciferase plasmids (t=10.01, 9.13, and 6149, P=0.010, P=0.012, and P＜0.001). ConclusionHsa_circ_0091579 is highly expressed in HCC cell lines and may play the role of oncogene by inhibiting miR-149, miR-490-5p, and miR-502-5p.

Regulatory mechanisms of the transcription and metabolism of hepatitis B virus covalently closed circular DNA and strategies for silencing and elimination / 临床肝胆病杂志

It is known that hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) persists in the nucleus of infected hepatocytes in the form of minichromosome and is difficult to target and eliminate. Studies on the mechanisms and strategies for persistent silencing or elimination of HBV cccDNA are the focus achieving for “functional cure” of chronic hepatitis B. This article introduces the current knowledge on the basic biological features of cccDNA, regulatory mechanisms of transcription and metabolism, and related host factors, with a focus on the potential pathways and strategies for cccDNA silencing or elimination.

Research advances in persistent hepatitis B virus infection and its mechanism / 临床肝胆病杂志

Hepatitis B virus (HBV) belongs to the Hepadnaviridae family and can cause acute and chronic hepatitis, liver cirrhosis, and even liver cancer in humans. Current antiviral drugs cannot completely eliminate HBV in liver cells and thus it is difficult to achieve a curative effect. In recent years, the mechanism of persistent HBV infection has attracted wide attention, which mainly involves host and virus. This article elaborates on the research advances in persistent HBV infection from the aspect of virus, including covalently closed circular DNA, HBV particles, and HBV components.

Hepatitis B virus core-related antigen: A promising new serum marker for hepatitis B virus / 临床肝胆病杂志

Hepatitis B virus core-related antigen (HBcrAg) is a new serum biomarker for hepatitis B virus (HBV) and is composed of several antigens encoded by the pre-C/C region gene of HBV, including HBcAg, HBeAg, and P22cr precursor protein. There is a poor correlation between HBcrAg and HBsAg and they cannot replace each other. Serum HBcrAg level can reflect the content and transcriptional activity of cccDNA in hepatocytes of patients with chronic hepatitis B, as well as the transcriptional activity of integrated HBV DNA. In addition, HBcrAg can be used to evaluate the antiviral effect of nucleos(t)ide analogues and pegylated interferon- and predict recurrence risk after withdrawal of nucleos(t)ide analogues and the development risk and recurrence of hepatocellular carcinoma after surgery. Therefore, serum HBcrAg is a promising new serum marker for HBV.

Application value of serum HBV RNA in clinical management of chronic hepatitis B / 临床肝胆病杂志

Viral hepatitis, liver cirrhosis, and hepatocellular carcinoma caused by hepatitis B virus (HBV) infection still threaten the health of people in China. Efficient antiviral therapy can significantly prevent the progression of liver diseases and reduce the incidence rate of end-stage liver disease, but it is very difficult to achieve clinical cure by current drugs, and nucleos(t)ide analogues may require long-term medication. New serological markers can be used for further optimization of antiviral regimens, among which serum HBV RNA is transcribed from covalently closed circular DNA (cccDNA) and is mainly derived from pregenomic RNA and its splice variants with no or partial reverse transcription. This article summarizes the sources and existing forms of serum HBV RNA, the influencing factors for serum HBV RNA level, the correlation between serum HBV RNA and other serum virological markers, and the correlation between serum HBV RNA and cccDNA in liver tissue, as well as the clinical significance of serum HBV RNA in monitoring antiviral efficacy and predicting the risk of viral rebound after drug withdrawal.

Clinical application of covalently closed circular DNA detection techniques / 临床肝胆病杂志

Covalently closed circular DNA (cccDNA) is the original template for hepatitis B virus (HBV) replication and is the basis of persistent HBV infection. Only when HBV cccDNA is completely eliminated can HBV be eradicated. Therefore, the detection of HBV cccDNA plays an important role in evaluating the antiviral outcome of chronic hepatitis B, assessing HBV clearance, clarifying occult HBV infection, studying the pathogenesis of hepatitis B, and developing new drugs, and HBV cccDNA detection techniques with high sensitivity and specificity are the basis for clinical application. With reference to related reports in literature and our own research work, this article reviews the development and clinical application of HBV cccDNA detection methods in recent years.

Research advances in therapeutic strategies against covalently closed circular DNA of hepatitis B virus / 临床肝胆病杂志

Chronic infection with hepatitis B virus (HBV) often leads to the development of liver cirrhosis and liver cancer, creating immense sociological, clinical, and economic burdens worldwide. Although current anti-HBV drugs can control the disease progression, they often fail to eliminate the virus because of the presence of covalently closed circular DNA (cccDNA) in the hepatocyte nucleus. We review the research advances in therapeutic strategies against cccDNA from the aspects of interfering with cccDNA synthesis, promoting cccDNA degradation, and silencing cccDNA. Targeting cccDNA is a promising approach that may lead to a cure of chronic HBV infection.

Detection of the covalently closed circular DNA in peripheral blood mononuclear cells of hepatitis B patients and its clinical significance / 中华传染病杂志

Objective To analyze the correlation between covalently closed circular DNA (cccDNA) in the peripheral blood mononuclear cells (PBMC) of hepatitis B virus (HBV)-infected patients and serum HBV DNA,hepatitis B surface antigen (HBsAg),hepatitis B e antigen (HBeAg) and liver histology of hepatitis B patients,and to explore the clinical significance of HBV cccDNA detection in PBMC.Methods One hundred and eight patients with chronic HBV infection were involved in this study.PBMC were extracted using density gradient centrifugation.HBV cccDNA in PBMC and serum HBV DNA were detected by real-time fluorescence quantitative polymerase chain reaction.HBsAg and HBeAg were detected by chemiluminescence immunoassay.Liver biopsy was conducted in 59 out of the 108 patients.Chi-square test was used to compare the categorical variables.Correlation analysis was used to compare quantitative variables.Nonparametric test was used to compare the non-normal distribution parameters.Results In the overall population,HBV cccDNA in PBMC was positive in 59 patients (54.6％).Eleven of the 15 patients with liver failure were found to be HBV cccDNA positive,which was significantly higher than that in the acute hepatitis B group (only 2 of the 8 patients were HBV cccDNA positive; x2 =4.960,P＜0.05).One hundred and eight patients were categorized into three groups according to their serum HBV DNA levels,with group A:＞5 lg copy/mL,group B:3-5 lg copy/mL and group C:＜3 lg copy/mL.The proportions of HBV cccDNA positivity in PBMC in three groups were 76.1％ (51/67),5/18 and 13.0％ (3/23),respectively.Comparing with patients with lower HBV DNA (group B and C),the proportion of HBV cccDNA positivity was higher in patients with higher HBV DNA (group A; x2=14.751,P＜0.05 and x2 =28.384,P＜0.05,resepectively).The HBV cccDNA quantitation in PBMC was positively correlated with the serum HBV DNA level and HBsAg quantification (r=0.554,P＜0.05 and r=0.497,P＜0.05,respectively).The proportion of HBV cccDNA positivity in PBMC of patients with liver histology ≥G2 and/or ≥S2 was significantly higher than that in patients with liver histology ＜ G2/S2 (x2 =9.159,P＜0.05).Conclusions HBV cccDNA exists in PBMC of hepatitis B patients.The HBV cccDNA quantitation in PBMC is positively correlated with the serum level of HBV DNA and HBsAg quantification,and is also associated with liver histology injury.

Significance and progress in detection of HBV cccDNA in peripheral blood mononuclear cells / 临床肝胆病杂志

Hepatitis B virus (HBV)covalently closed circular DNA (cccDNA)is an intermediate replicative episome which is stable and not easy to eliminate.cccDNA serves as the template for HBV replication and plays an important role in persistence of HBV infection in hep-atocytes,and it is also a biomarker of HBV activity.Studies suggested that HBV has extrahepatic infectivity and cccDNA could be detected in peripheral blood mononuclear cells (PBMC).The predictive values of HBV cccDNA in PBMC for HBV recurrence after liver transplanta-tion and antiviral therapy and mother-to -child transmission of HBV in patients with hepatitis B,as well as the possible mechanism of cccDNA involved in extrahepatic HBV infection,are summarized.Therefore,the detection of HBV cccDNA is of great significance for the study and treatment of HBV.

Efficacy of sequential add-on pegylated interferon α-2a in combination with adefovir dipivoxil in chronic hepatitis B patients with low serum HBeAg titer / 中华传染病杂志

Objective To investigate the efficacy of sequential add-on of pegylated interferon α-2a (PEGIFN-α-2a) for 48 weeks in chronic hepatitis B (CHB) patients with low serum hepatitis B e antigen (HBeAg) titer after long term adefovir dipivoxil (ADV) monotherapy.Methods This was a randomized,open and prospective clinical trial.Patients who had been treated with ADV for 72 to 144 weeks,with undetectable serum hepatitis B virus (HBV) DNA level,low HBeAg titer (5 S/CO＜ HBeAg＜50 S/CO) and serum hepatitis B surface antigen (HBsAg) ＜5000 IU/mL were included.The patients were categorized into ADV monotherapy group and ADV plus PEGIFN-a-2a combination therapy group by random number table.Patients in ADV group continued ADV monotherapy and patients in combination therapy group added PEGIFN-α-2a to ADV for 48 weeks.After the treatment,efficacy of the two therapies were assessed by comparing the reduction of serum HBeAg reduction,HBeAg loss rate,HBeAg seroconversion rate,and reduction of intrahepatic HBV DNA and HBV covalently closed circular DNA (cccDNA).Pre-and post-treatment results were compared by paired samples t test.Comparison between groups was performed using indepedent samples t test.Comparison of rates between groups was performed using x2 test.Results The trial enrolled 55 CHB patients,and there were 27 patients in ADV monotherapy group,28 patients in combination therapy group.Baseline characteristics including age distribution,sex ratio,alanine aminotransferase (ALT),aspartate aminotransferase (AST),total bilirubin (TBil),serum HBeAg and HBsAg,hepatic HBV DNA and HBV cccDNA were all comparable (all P＞0.05).Twenty-five patients in ADV monotherapy group and 26 patients in combination therapy group completed 48 weeks treatment.HBeAg loss rates and seroconversion rates of combination therapy group were higher than those of ADV monotherapy group (x2 =5.38 and 4.69,respectively,both P＜0.05).HBeAg titers of both groups were significantly lower than those of baseline (t=8.43 and 8.50,respectively,both P＜0.05).The HBeAg titer of combination therapy group was lower than that of monotherapy group (t=5.60,P＜ 0.01).HBV DNA and HBV cccDNA in liver tissue of combination therapy group was (6.934±0.52) lg IU/mg and (5.63±0.54) lg IU/mg post-treatment,respectively,which were both lower than baseline (t＝7.12.6.67,respectively,both P＜0.01).HBV DNA in liver tissue of monotherapy group was (7.09＝0.43) lg IU/mg post-treatment,which was lower than baseline (t=2.67,P=0.02).After treatment,HBV cccDNA in liver tissue of combination therapy group was lower than that of monotherapy group (t =2.87,P=0.00).Conclusions Compared with ADV monotherapy,sequential add-on of PEGIFN-a-2a in combination with ADV can achieve higher serum HBeAg loss rate and seroconversion rate and facilitate the clearance of hepatic HBV DNA and HBV cccDNA in CHB patients with low HBeAg titer after long-term ADV monotherapy.

Comparison of drug resistance mutations associated with hepatitis B virus covalently closed circular DNA and relaxed circular DNA / 中华传染病杂志

Objective To detect nucleos(t)ide-resistance mutations in hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) isolated from hepatocytes of patients with chronic HBV infection and to analyze the correlation between the mutations found in cccDNA and relaxed circular DNA (rcDNA). MethodsForty patients with chronic HBV infection were investigated. Preoperation serum samples and non-tumor liver tissues were collected.Intrahepatic HBV cccDNA and rcDNA were selectively extracted by co-precipitation of sodium dodecyl sulphate-protein and QIAamp DNA Mini Kit, and further purification with plasmid-safe ATP-dependent DNase (PSAD).Thereafter,cccDNA were amplified by selective polymerase chain reaction (PCR) or nested PCR using the primers spanning both the two gaps in HBV genome and covering the common mutations associated with nucleoside analogues resistance (rt169- rt250).Intrahepatic HBV rcDNA and pre-operation serum HBV rcDNA were also extracted and then amplified by PCR.The PCR products were then purified and sequenced.Results Among the 40 patients,intrahepatic HBV cccDNA were detected in 31 patients,and HBV rcDNA were detected in liver samples of 35 patients and pre-operation serum samples of 21 patients. The PCR products amplified from these samples were all successfully sequenced.rtM204Ⅰ mutation was detected in intracellular HBV cccDNA,rcDNA and serum HBV rcDNA in 2 patients.Both rtM204Ⅰ and rtQ215H were detected in intrahepatic HBV cccDNA and rcDNA in 2 patients,while no corresponding mutation was observed in serum HBV rcDNA of these two patients.Three variants including rtM204V,rtM204V and rtV173L-rtL180M-rtM204V were detected in serum HBV rcDNA in 3 patients,while corresponding mutants were not detected in intracellular HBV cccDNA or rcDNA of these patients.Condsions The results suggest that antiviral nucleos(t) ide resistance mutations can be found in HBV cccDNA in chronic hepatitis B patients. The dominant resistant mutation found in intrahepatic HBV cccDNA/rcDNA may be different from that in serum HBV rcDNA.

The relationship between function of dendritic cells and hepatitis B virus covalently closed circular DNA in the peripheral blood mononuclear cells of patients with chronic hepatitis B / 中华传染病杂志

Objective To investigate the relationship between the maturity and function of dendritic cells (DC) and hepatitis B virus covalently closed circular DNA (HBV cccDNA) load in the peripheral blood mononuclear cells (PBMC)/monocyte-derived DC in patients with chronic hepatitis B (CHB). Methods The peripheral blood samples were collected from 29 patients with CHB and 10healthy controls. PBMC were isolated freshly and induced with granulocyte/macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4). A large amount of DC were harvested after seven days of culture. The expressions of CD209, CD80, CD86, human leucocyte antigen (HLA)-DR and CD1a of DC were analyzed by flow cytometry. The HBV cccDNA load in PBMC and DC were measured by real-time polymerase chain reaction (PCR). The interleukin-12 (IL-12) level in the culture supernatant of DC was determined by enzyme linked immunosorbent assay (ELISA). The effects on T lymphocyte proliferation induced by DC were tested by mixed lymphocyte reaction (MLR). The data was compared by t test and analysis of variance. Results HBV cccDNA could be detected in PBMC from 16 patients, but not in DC from all 29 patients. HBV cccDNA load was all negatively correlated with the expressions of CD209 (r= -0. 793, P＜0.01), CD80 (r= -0. 581,P＜0.05), CD86 (r=-0. 698, P＜0.01), HLA-DR (r=-0. 817, P＜0.01), CD1a (r=-0. 734, P＜0.01), IL-12 level (r=-0. 632, P＜0.05) and allogenic T lymphocyte proliferation induced by DC (r=-0. 617, P＜0.05). The expressions of CD209, CD80, CD86, CD1a and HLA-DR on DC,IL-12 level in culture supernatant of DC and the allogenic T lymphocyte proliferation induced by DC in patients with positive PBMC HBV cccDNA were all significantly lower compared to those in healthy controls, and the changes of the parameters mentioned above were greater in PBMC HBV cccDNA positive patients than those in PBMC HBV cccDNA negative patients (P ＜ 0. 05 or P ＜ 0. 01).Conclusions The function and maturity of DC are impaired in CHB patients. HBV cccDNA can be detected in PBMC from CHB patients. Moreover, the higher PBMC HBV cccDNA is, the worse DC function and maturity are, which could be one of the important mechanisms of HBV persistent infection.

The study on the rolling circle amplification for detecting hepatitis B virus covalently closed circular DNA / 中华传染病杂志

Objective To set up the rolling circle amplification (RCA) system for detecting hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), and to evaluate the specificity and sensitivity of this system. Methods Plasmids containing full-length of wild-type HBV genome were treated with restriction enzyme and T4 DNA ligase, and then were concentrated. The DNA fragments were recovered by the nucleic acid purification kit and severed as standard HBV cccDNA. Total DNA was extracted from hepatic tissues of seven chronic hepatitis B patients. RCA method was used to amplify genomes from tissue samples. Standard HBV cccDNA, 3.2 kb liner HBV DNA, normal hepatic tissue samples and 15 serum samples of patients with chronic HBV infection were used as controls to determine the specificity of RCA. Ten-fold serial dilutions of standard HBV cccDNA were used for determining the sensitivity. Results The standard HBV cccDNA was successfully constructed and could be detected by RCA method. HBV cccDNA could be amplified from 2 mg hepatic tissue samples at least of HBV infected patients, and could be detected as low as 1 ×102 copy/μL. cccDNA was not detected in 3.2 kb liner HBV DNA, normal hepatic tissue samples and 15 serum samples of chronic HBV infected patients. Conclusion RCA method can be used for rapid and simple detection of HBV cccDNA with high specificity and sensitivity.

Inhibition effect of total glucosides of Picrorhiza on replication of hepatitis B virus in vitro / 中华传染病杂志

Ohjective To observe the inhibition effect of total glucosides of Picrorhiza on hepatitis B virus covalently closed circular DNA (HBV cccDNA) in HepG 2.2.15 cell line. Methods HepG 2.2.15 cells were incubated with culture medium containing 50 mg/L of picrosides or 5 mg/L of adefovir dipivoxil for 2 or 5 days. HBV DNA in the supernatant, intracellular cccDNA, relaxed circular DNA (rcDNA) and pregenomic RNA (pgRNA) were quantified by specific real-time polymerase chain reaction (RT-PCR) and inhibition rates were calculated. The means were compared by t test. Results After treated with picrosides for 2 and 5 days, the inhibition rates of HBV DNA in thesupernatant were 49. 74% (t=4.723, P<0.05) and 79.48% (t = 7.512, P<0.05), respectively. The inhibition rates of intracellular cccDNA were 43.55% (t = 5.216, P<0.05) and 56.43% (t=7.262, P<0.05), respectively, while those of intracellular rcDNA were 43.39% (t=4.137, P<0.05) and 63.86% (t=7.861, P<0.05), respectively, and those of intracellular pgRNA were 54.72% (t=4.532, P<0.05) and 56.08% (t=4.833, P<0.05), respectively. Comparatively, after treatment with adefovir dipivoxil for 2 and 5 days, the inhibition rates of HBV DNA in the supernatant were 25.56% (t=2.874, P<0.05) and 92.44% (t =10.276, P<0.05), respectively. Those of cccDNA were 18.54% (t=2.736, P<0.05) and 47.19% (t=6.852, P<0.05), respectively. Those of rcDNA were 21. 20% (t=3.206, P<0.05) and 71.47% (t=8.332, P<0.05), respectively, pgRNA were 11.14% (t=1.761, P>0.05) and 37.61%(t=3.632, P<0.05) respectively in HepG2.2.15 cells. Conclusions Pierosides may inhibit the replication cycle of HBV, including the formation of cccDNA in HepG 2.2.15 cells. The mechanism of pierosides on cccDNA may differ from adefovir dipivoxil's due to its earlier inhibition time phase.

Detection of hepatitis B virus covalently closed circular DNA in serum of patients with hepatitis B / 中华传染病杂志

Objective To determine whether hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) could be detected in serum of patients with hepatitis B and evaluate the related factors and clinical significances. Methods Fifty-seven patients, including 26 with mild chronic hepatitis B (CHB) and 31 with severe hepatitis B (SHB) were enrolled. Prothrombin time (PT), hepatic biochemical indexes, serum markers of hepatitis virus, serum total HBV DNA and HBV cccDNA of every patient were detected after hospitalization. Factors associated with the detection rate of serum HBV cccDNA were analyzed using Logistic stepwise regression. Results Serum HBV cccDNA was detected in 13 patients with SHB and 1 with mild CHB, and serum levels of HBV cccDNA were varying from 1.25 × 103 to 4. 88 × 104 copy/mL. The detection rates were significantly different between the two groups (P=0. 0014). The sensitivity and specificity of SHB diagnose by serum HBV cccDNA detection were 41.94% and 96.15 %, respectively. Logistic regression analysis showed that the detection rate of serum HBV cccDNA was associated with PT (X2 = 7. 2192, P= 0. 0072), while not associated with age, sex, total serum HBV DNA, total bilirubin or alanine aminotransferase (ALT). Conclusion Serum HBV cccDNA could be detected in some of the patients with SHB, whic hmay be considered as one of the diagnostic indexes for SHB,

Quantitative detection of hepatitis B virus cccDNA in chronic hepatitis B patients / 中华检验医学杂志

Objective To establish a method for detecting HBV cccDNA in hepatocytes of chronic hepatitis B patients.Method 21 liver biopsies from the hepatic operation patients in the hospital of jiangsu province,concluding 19 HBV chronic infected patients (10 HBeAg positive patients and 9 HBeAg negative patients) and 4 uninfected patients,HBV DNA(+) serum of hepatitis B patients was thought as rcDNA.To use proteinase K to release HBV cccDNA and genomic DNA,then divide the cell lysis solution into two parts,one for detecting HBV cccDNA,the other for detecting the number of ?-Globin as internal control. Nucleic acid for detecting HBV cccDNA extracted by phenol-chloroform was digested by plasmid-safe ATP dependent DNase which was applied to digest the single strand DNA in rcDNA and ssDNA,then was quantitated by the primers spanning across the nick and SYBR Green Ⅰ dye.The specifity of PCR production was confirmed by the sequence analysis and rcDNA comparison.The significance of the difference of HBV cccDNA level between HBeAg(+) and HBeAg(-) group was analyzed by two group t test.Results The agarose gelelectrophoresis showed the molecular weight of the PCR production was about 350bp.The coincidence rate of PCR production and goal fragement was nearly 99% by sequence analysis.The result of PCR detection of rcDNA group was negative.The positive rate of HBV cccDNA of liver biopsies of HBeAg (+) patients detected by this method was 100%,the level of HBV cccDNA in the liver biopsies of HBeAg (+) patients was higher than HBeAb(+) patients.Conclusions The specificity of the method is proved by agarose electrophoresis,gene sequencing of the PCR product and rcDNA comparison.The quantitative method that use SYBR Green Ⅰ dye and ?-Globin as internal control is more specific,sensitive and economical,and more suitable for clinical purpose.