| Hepatitis B virus (HBV) infection is a major public health problem , and worldwide more than 350 million people are chronic carriers of the virus. After infected HBV, the majority of the patients is chronic infection and become chronic hepatitis B gradually, so the therapy to control active inflammation is very important. Expect IFN-a, Lamivudine is another antiviral agent that can inhibit the replication of HBV effectively. YMDD motif of HBV is the combining site of lamivu-dine. While long-term therapy will induce HBV DNA polymerase gene mutations and lamivudine resistance. So the outcome of lamivudine long-term therapy, the characters of HBV DNA polymerase gene mutations induced by lamivudine, the relationship of mutations and genotypes , and the countermeasures after mutations happened have become recent research points. The aim of this file is to research the outcome of one-year lamivudine therapy, the characters of HBV DNA polymerase gene mutations, the relationship of mutations and genotypes, and the factors affecting mutations.Materials & MethodsMaterials1. Subjects and serum samples collected:Test group: There are 33 patients with chronic hepatitis B, 22 male, 11 female, mean age is 31.79 years. All the patients take lami-vudine orally, lOOmg/d, beyond one year. Serum samples from baseline, 6 months and 12 months after therapy were collected and stored at -70^ for further detection.Control group: There are 20 asymptomatic carriers, 12 male, 8 female, mean age is 29. 5 years. The serum samples were collected and stores at -70*^0 for further detection when they were enrolled.2. Agents and equipments;(1) Primers: According to the sequence of HBV DNA polymer-ase gene (AF100309) , we designed three primers with DNA STAR software, named HBV381, HBV840, HBV801 respectively. The sequences are 5'-TGCGGCGTTTTATCATCTTCCT-3', 5'-GTTTAAATG-TATACCCAAAGAC-3' and 5'-CAGCGGCATAAAGGGACTCAAG-3'. And two pair hemi- nested PCR primers were formed: HBV381/HBV 840 ^ HBV381/HBV801. The primer of sequencing reaction is HBV381.(2) Wizard PCR Preps DNA Purification System and ABI PRISM Big Dye TM Terminator Cycle Sequencing Ready Reaction Kits.(3) ABI Gene Amp PCR System 2400.(4) ABI Gene Amp PCR System 9600.(5) ABI377 automatic sequencer. Methods1. Extraction of HBV DNA.2. Hemi-nested PCR:(1) First round amplification: Each reaction is 50ul, and the size of product is 453bp. The thermal cycling condition is 94℃ 45sec, 50℃30sec, 72℃1min30sec, 30 cycles.(2) Second round amplification: Each reaction is 50ul,and the size of product is 415bp. The thermal cycling condition is 94℃45sec, 55℃30sec, 72℃1 min, 30 cycles.3. Purification, sequencing reaction, precipitation and automatic sequencing.4. Analysis data.(1) Summarize the outcome of lamivudine therapy, observe the time of YMDD mutation development, and analysis the influence factors of outcome and mutation by SAS software.(2) Do phylogenetic analysis of part P gene of HBV DNA, and compare the difference of nucleotide and amino acid as well as draw the classification of mutation by CLUSTAL V method of DNA STAR software.(3 ) Analysis the difference of protein secondary structure of HBV DNA P gene encoded before and after mutation by DNAclub and DNAsis software.Results1. The outcome of lamivudine therapy: According to ALT ULN baseline group were classified to four groups:>5 x ULN,2-5 x ULN, 1-2 x ULN ,< 1ULN, as tab. 2. There are not different in improving ALT normalization in four individual groups. After 12 months of therapy , the rate of HBeAg loss increases linearly with ALT level of baseline increasing, the rate is 72.41% ; but the rate of HBeAg serocon-version decreases linearly with ALT level of baseline increasing, the rate is 13.79% ; and no HBsAg loss or HBsAg seroconversion was de-tected. YMDD mutation has not been detected from serum specimens of 6 months after therapy, and two YMDD mutations have been detected from serum specimens o...
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