A Study Of HBV Reverse Transcriptase Expression In PBMCs Of Viral Hepatitis B Patients

Objective To explore the clinical correlation of HBV reverse transcripts expression in peripheral blood mononuclear cells (PBMCs ) and hepatitis B clinical progression. Method We selected 70 cases patients with hepatitis B virus infection. Among the 70 cases, 19 acute hepatitis B, 12 slight chronic hepatitis B, 13 moderate chronic hepatitis B, 11 severe chronic hepatitis B and 15 cirrhosis respectively. There had been other 10 cases healthy volunteers as interferon group. The PBMCs were isolated from these patients' blood samples separately and the total RNA was extracted from PBMCs. DNase I without RNase was used to eliminate the remained DNA genome and then reverse transcript cDNA. P gene primers were selected for reverse transcription-polymerize chain reaction (RT-PCR) technology to determine HBV mRNA for explores HBV reverse transcriptase expression. One of the positive samples was cloned and sequenced. The purified product of 379bp long fragment was ligased to T-Easy vector , and then transformed into the E.coli DH5 a competent cells. The recombinant plasmids were confirmed by endonucleotidase analysis after white-blue clone selection. The plasmids from the positive clone were sequenced. The gained sequences were submitted to GenBank databases through Internet for homology analysis of nucleotide sequences with known sequences. Results The result of being sequenced was 379bp,which been compared with the standard HBV adw2 genotype, there were only 24 different nucleotides among 379bp.So the result was credibility. None of the healthy volunteers group was positive case. A lower positive rate of PBMCs HBV mRNA was detected in acute hepatitis B group: 5.3% (1/19), The total positive rate of chronic hepatitis B group was 33.3% (12/36) ; Higher positive rates were found in chronic hepatitis B group than in acute group, they were 25.9% (3/12 ) , 23.1% ( 3/13) and 54.5% (6/11) .In cirrhosis group the positive rate was 26.7%9 (4/15 ). The positive rate was significantly different between the acute and chronic hepatitis B patients groups (P<0.05) . The positive rate of thesevere chronic hepatitis B group (including those who had been cirrhosis patients and then suffer severe hepatitis) was higher as compared with the chronic hepatitis patients of slight and moderate groups (P<0.05) . But the positive rate was not significantly different between the chronic hepatitis B and the cirrhosis groups. Besides, the positive rate of HBV mRNA in PBMCs was not correlated with the positive rate of HBeAg and HVBDNA in serum (r =0.164, P>0.05; r=0.39, P> 0.05 respectively) .But the positive rate of PBMCs HBV mRNA was correlated with that of hepatitis B patients with family history patients (37.0%; 16.3%. r=0.229, P <0.05) .Conclusion HBV can replicate in PBMCs independently and become the resource of reactivation; expression of HBV reverse transcript mRNA in PBMCs may be related to the chronicity and seriousity of hepatitis B, and it has a close relationship with family history in hepatitis B patients; To quantify PBMCs HBV mRNA my be useful in the evaluation of early disease progression.