| The first section: Analysis of HBV reverse transcriptase gene sequence characteristics in Entecavir -resistant patientObjective:To study the nucleotide substitution sites and patterns of the reverse transcriptase(RT)domain of HBV polymerase with entecavir -resistant patient under NAs therapy.Methods: Between April 2006 and February 2011, the objcet were selected from patients with chronic hepatitis B who were treated in the First Affiliated Hospital of Soochow University. Amino acid sequence of HBV RT domain which amplified by PCR and then directly sequenced was analysized using Chromas 2.0 software.Results: 29 ETV- resistant patients were deteced, 6(26.09%) patients with viral breakthorugh under LAM therapy, 23(73.90%) patients with viral breakthorugh under LAM-ETV sequential therapy. The entecavir resistance patterns of HBV RT domain includ rtT184,rtM250 and rtS202G, respectively for 15 cases(62.07%),8cases(27.58%) and 6 cases(26.09%), presented as T184V/A/G /I/S /M,M250I/L/V and rtS202G. Meanwhile, some new amino acid substitution,such as A165C,S168C,P170S,S172M,V175I, V183G, V207M/I,V214A,A222T,S223A,I224V,F226T/C,N227T,L229M,T237P,N248H,I254M,V256S were detected.Conclusion: ETV resistance was induced by LAM or LAM-ETV sequential therapy. The resistance patterns of HBV RT domain were LAM resistance(mutations of rtM204I/V plus rtL180M or not) in combination with ETV resistance(mutations of rtT184,rtM250 or rtS202G), presented as T184I/G/S,rtS202G and M250L/V. The second section: Adefovir plus entecavir or plus lamivudine as rescue therapies for chronic hepatitis Bpatients who develop resistance to entecavir Objective: To investigate the efficacy and safety of Adefovir(10mg/d) plus Entecavir(0.5mg/d) or plus Lamivudine(100mg/d) in entecavir-resistant Chronic hepatitis B patients.Methods: Between April 2006 and February 2011, the objcet were selected from patients with NAs experienced and viral DNA breakthorugh who were treated in the First Affiliated Hospital of Soochow University.The 29 cases with ETV- resistant were divided into group A and group B according to different therapies. The group A were treated with ADV10mg/d plus LAM100mg/d, and the group B treated with ADV10mg/d plus ETV0.5mg/d. The serum HBV DNA (measured via real-time polymerase chain reaction), liver and renal function and HBV serum mark at based line and 12, 24, 48, 96-week were detected. There 3 cases with the decreases of HBV DNA<2log10copies/ml in group A who finished 24 weeks, switched to ADV10mg/d plus ETV0.5mg/d and continue to monitoring and follow up.Results: There were 21and 8 patients in group A and B, respectively.The time of therapy was 69weeks in average,the range 8~92weeks. The mean decreases of HBV DNA level were 2.87±0.98 vs1.62±1.01 log10copies/ml (p=0.033),and the rates of HBV DNA was reduced to the level under detection were 2/19(10.53%)vs 1/7(14.29%) (p=0.096)at 24-week among in groupA and B, respectively.The mean decreases of HBV DNA level were 1.0±0.89vs 1.06±1.25 log10copies/ml(p=0.096),the cases of HBV DNA was reduced to the level under detection were 8 and 2 at 48-week among in groupA and B, respectively. The cases of HBeAg seroconversion were 3 cases. There were 3 and 5 cases with the decreases of HBV DNA<2log10copies/ml at 24-week among in groupA and B. The rates of HBV DNA was reduced to the level under detection were 80%(4/5) after prolong the treatment to 48 weeks among 5 patients(2 cases in group A or B)with downtrend of HBV DNA.The serum HBV DNA level decreases 2.84±0.65 log10copies/ml at 24-weeks,and obtain the complete virological response among 3 patients in group B with no downtrend of HBV DNA. Besides only one patients in groupA with transient proteinuria,there was not severity adverse events.Conclusion: The Adefovir plus entecavir or plus lamivudine could as the rescue therapies for CHB patients who develop resistance to Entecavir,but the efficary was limit.Prolong the treatment or change the plan according the respond will enhance the efficary.The third section: Amplification and cloning the a full-length HBV genome by polymerase chain reaction(PCR),and construction of HBV Gene Eukaryotic Cell Expression Vector.Objective: To improve the method of amplification and cloning of a full-length HBV genome by polymerase chain reaction(PCR). Amplification and analysis of whole HBV genome sequence with entecavir- resistant To construct a mammalian HBV recombinan tvector carrying entire HBV genome.Methods: Optimization the extract of sample and the of PCR proposal for improving the technique PCR for amplification the whole HBV genome . The 19 cases of CHB patients with Entecavir-resistant were amplified by improvement PCR,and analyzed the differences of amino acid , variation type and genotypes between the sequence and standard in GenBank by Chromas2.0,DNAMAN software.Using molecular subclone technique, pcDNA was prepared by insertion of the entire HBV genoma in to the Sca I site.Results: The 3.2 kb full-length HBV genome was successfully obtained by this method,the min serum HBV DNA load of sample is 3.95log10cop/ml.In the full- length HBV genome ,besides the commom mutations site RT domain,there were 18.75%(3/16)and 31.25%(5/16) deletion site Pre1 and PreS2 region was observed respectively.The mutation of pG 83A in C region was observed was 5 cases (31.25%). There were mutations correlatd with T, B, CTL cell epitope variation ,or were detected as coincide variations in S and RT domains.The plasmid pcDNA-HBV3.2 containing the 3.2 kb full-length HBV genome was successfully constructed.Conclusion: The cloning of full-length HBV genom from serum samples and HBV Gene Eukaryotic Cell Expression Vector are successfully established,which could serve to further study the relationship between HBV mutation and pathogenesis. Besides the commom mutations site RT domain, The HBV genome with entecavir- resistant could develop the deletion site Pre1 ,PreS2 and C region, or coincide variations in S and RT domains. |
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