| This research was composed of two parts: I . Semi-Quantitative detection of hepatitis B virus cccDNA in liver tissues;II. To detect and analyze hepatitis B virus cccDNA in the sera and liver tissues of patients who were HBV infected.I .Semi-Quantitative detection of hepatitis B virus cccDNA in liver tissuesObjective To establish a method for semi-quantitative detection of hepatitis B virus cccDNA in liver tissue.Methods 40 liver tissues which involved cancerous tissues and pericancer tissues of 20 patients with hepatocellular carcinoma were investigated .Total DNA were extracted from the tissues with proteinase K and phenol-chloroform. The extraction products were further purified by Plasmid-Safe ATP-dependent Dnase to degrade linear double-stranded DNA, closed-circular and linear single-stranded DNA. HBV cccDNA were detected by PCR with selective primer set .Results cccDNA were detectable in liver tissues, and HBV DNA of liver tissues which were digested were lower than that were undigested(0.100[negative~1.960]and 0.481[negative~3.160];P=0.000);cccDNA of liver tissues which were digested were lower than that were undigested (0.130[negative~1.770] and 0.421 [negative~2.360];P=0.004);cccDNA of liver tissues which were digested by Plasmid-Safe? ATP-dependent DNase and detected by different primer sets were positive correlation ( r=0.844,P=0.000 ) . Conclusions This method is convenient,relative highly specific.It can be semi-quantitative detect cccDNA in the liver tissue.II. To detect and analyze hepatitis B virus cccDNA in the sera and liver tissues of patients who were HBV infected.Objective To study the existing of cccDNA in the sera and liver tissues of patients who were infected by HBV. Methods Based on the difference of cccDNA and rcDNA ,cccDNA in the sera of 115 patients who were infected by HBV and in the liver tissues of 20 HCC patients were detected by PCR and were verified by agarose gel electrophoresis, gray scales were analyzed. Results In 115 patients who were infected by HBV ,the sera cccDNA levels were found to correlate with sera HBV DNA levels (r=0.702,P=0.000);HBeAg-positive patients had higher median serumcccDNA levels than HBeAg-negative patients (0.465 [negative~2.050] and 0.080[negative~1.760];P=0.026);the sera cccDNA levels were found to correlate with ALT (r=0.347,P=0.000) . In 20 HCC patients, HBV DNA was detected in 17(85%) cancerous tissues and the median was 0.231;HBV DNA was detected in 17(85%) pericancer tissues and the median was 0.829;there was no significant difference between the cancerous tissues and the pericancer tissues (P=0.701) . cccDNA was detected in 9(45%) the cancerous tissues and the median was 0.100;cccDNA was detected in 13(65%) the pericancer tissues and the median was 0.214;there was no significant difference between the cancerous tissues and the pericancer tissues (P=0.213) .There was a positive correlation between the cccDNA and HBV DNA in the liver tissues (r=0.778,P=0.000) . The sera HBV DNA levels were found to correlate with cancerous tissues HBV DNA levels (r=0.789,P=0.000) and the pericancer tissues HBV DNA levels (r=0.589,P=0.021) . The sera cccDNA levels were found to correlate with cancerous tissues cccDNA levels ( r=0.566,P=0.002 ) and the pericancer tissues cccDNA levels (r=0.465? P=0.036). The cccDNA levels of the cancerous tissues were higher than the cccDNA levels of the sera(0.100[negative~1.770] and 0.010[negative~0.560];P=0.030). The cccDNA levels of the pericancer tissues were higher than the cccDNA levels of the sera(0.214[negative~1.390] and 0.010[negative~0.560];P=0.001 ) . Conclusions cccDNA can be detected in the sera . It is possible that the lysis of infected hepatocytes are one of the source of serum cccDNA. The cancerous tissues and the pericancer tissues of HCC patients who were infected by HBV exist cccDNA. Detection of cccDNA in the liver tissues may provide a guidance of anti-HBV therapy and can help to select the time of ceasing anti-HBV therapy.
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