郑州工业高专三届新生乙型肝炎病毒感染分析

为了解高校新生乙型肝炎病毒感染状况,对我校新生进行乙型肝炎病毒表面抗原检测。结果显示三届新生乙型肝炎病毒染率为7.6％,低于全国人群平均感染水平;男生感染率高于女生;乙型肝炎病毒感染地区分布南方高于北方;乙型肝炎病毒疫苗对健康人群有很好的保护作用。必须采取综合措施才能减少乙型肝炎在校内传播。

Epigallocatechin gallate inhibits HBV DNA synthesis in a viral replication-inducible cell line

AIM:To analyze the antiviral mechanism of Epigallocatechin gallate(EGCG)against hepatitis B virus(HBV) replication.METHODS:In this research,the HBV-replicating cell line HepG2.117 was used to investigate the antiviral mechanism of EGCG.Cytotoxicity of EGCG was analyzed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.Hepatitis B virus e antigen(HBeAg)and hepatitis B virus surface antigen(HBsAg)in the supernatant were detected by enzyme-linked immunosorbent assay.Precore mRNA and pregenomic RNA(pgRNA) levels were determined by semi-quantitative reverse transcription polymerase chain reaction(PCR)assay.The effect of EGCG on HBV core promoter activity was measured by dual luciferase reporter assay.HBV covalently closed circular DNA and replicative intermediates of DNA were quantified by real-time PCR assay.RESULTS:When HepG2.117 cells were grown in the presence of EGCG,the expression of HBeAg was suppressed,however,the expression of HBsAg was not affected.HBV precore mRNA level was also downregulated by EGCG,while the transcription of precore mRNA was not impaired.The synthesis of both HBV covalently closed circular DNA and replicative intermediates of DNA were reduced by EGCG treatment to a similar extent,however,HBV pgRNA transcripted from chromosome-integrated HBV genome was not affected by EGCG treatment,indicating that EGCG targets only replicative intermediates of DNA synthesis.CONCLUSION:In HepG2.117 cells,EGCG inhibits HBV replication by impairing HBV replicative intermediates of DNA synthesis and such inhibition results in reduced production of HBV covalently closed circular DNA.

Development of HBsAg-Binding Aptamers that bind HepG2.2.15 cells via HBV surface antigen

Hepatitis B virus surface antigen （HBsAg）, a specific antigen on the membrane of Hepatitis B virus （HBV）-infected cells, provides a perfect target for therapeutic drugs. The development of reagents with high affinity and specificity to the HBsAg is of great significance to the early-stage diagnosis and treatment of HBV infection. Herein, we report the selection of RNA aptamers that can specifically bind to HBsAg protein and HBsAg-positive hepatocytes. One high affinity aptamer, HBs-A22, was isolated from an initial 115 met library of -1.1 ×10^15 random-sequence RNA molecules using the SELEX procedure. The selected aptamer HBs-A22 bound specifically to hepatoma cell line HepG2.2.15 that expresses HBsAg but did not bind to HBsAg-devoid HepG2 cells. This is the first reported RNA aptamer which could bind to a HBV specific antigen. This newly isolated aptamer could be modified to deliver imaging, diagnostic, and therapeutic agents targeted at HBV-infected cells.

Inhibition of hepatitis B virus surface antigen expression by small hairpin RNA in vitro

AIM: To explore the anti-hepatitis B virus effect of RNA interference (RNAi) using small hairpin RNA (shRNA)expression vector.METHODS: Hepatitis B virus surface antigen green fluorescent protein (HBs-GFP) fusion vector and shRNA expression vectors were constructed and cotransfected transiently into HepG2 cells. mRNAs extracted from HepG2 cells were detected by real-time PCR. Fluorescence of HBs-GFP protein was detected by fluorescence-activated cell sorting (FACS). The effective shRNA expression vector was transfected into HepG2.2.15 cells. HBsAg and HBeAg in HepG2.2.15 cells were analyzed by radioimmunoassay (RIA) method.RESULTS: FACS revealed that shRNA targeting at HBsAg reduced the GFP signal by 56% compared to the control.Real-time PCR showed that HBs-GFP mRNA extracted from HepG2 cells cotransfected with pAVU6+27 and HBs-GFP expression plasmids decreased by 90% compared to the empty vector control. The expressions of HBsAg and HBeAg were also inhibited by 43% and 64%, respectively.CONCLUSION: RNAi using shRNA expression vector can inhibit the expression of HBsAg, providing a fresh approach to screening the efficient small interfering RNAs (siRNAs).

Effect of lamivudinein in BeAg-positive chronic hepatitis B: Discordant effect on HBeAg and HBV DNA according to pretreatment ALT level

AM: To clarify differences in antiviral effect of the drug in patients with different ALT levels, we examined the changes in HBV markers in patients with high or low ALT levels with or without lamivudine treatment. METHODS: Thirty-seven HBeAg-positive patients were studied. Ten patients with ALT levels higher than 200 IU/L (group 1) and 8 patients with ALT below 200 IU/L (group 2) were treated orally with 100 mg/d of lamivudine. As untreated control, 9 patients with ALT above 200 IU/L (group 3) and 10 patients with ALT below 200 IU/L (group 4) were examined. ALT level, HBeAg/HBeAb status, and HBV DNA level were examined monthly for 11.9±0.4 mo. RESULTS: The ALT level normalized in all 10 patients of group 1, 7/8 of group 2, 4/9 of group 3, and 1/10 of group 4 within 6 mo (groups 1 vs2, P= NS; groups 1 vs 3, P= 0.002; groups 1 vs4, P<0.0001). HBV DNA fell below the detection limit in all 10 patients of group 1, 7/8 of group 2, 0/9 of group 3, and 0/10 of group 4 within 6 mo (groups 1 vs 2, P - NS). HBeAg became seronegative in 7/10 patients of group 1, 1/8 of group 2, 3/9 of group 3, and 0/10 of group 4 within 12 mo (groups 1 vs2, P= 0.02; groups 1 vs 3, P= NS). CONCLUSION: Our data suggest that HBeAg-positive patients with higher ALT levels can be considered good candidates for lamivudine therapy, probably because lamivudine accelerates the natural seroconversion of HBeAg, accompanied by HBV DNA loss, in these patients.

Toll-like receptor 4 plays an anti-HBV role in a murine model of acute hepatitis B virus expression

AIM： Toll-like receptor 4 （TLR4） has been shown to be important for bacterial infection, especially to lipopolysaccharide signaling. Its possible role in HBV infection is studied in the present study. MATERIALS AND METHODS： pHBV3.6 plasmid, containing full-length HBV genome was used in the murine model of acute HBV expression by hydrodynamics in vivo transfection. TLR4 normal or mutant mouse strain was compared to investigate the possible role of TLR4 in acute HBV expression. RESULTS： After pHBV3.6 injection, the infiltrating leukocytes expressed TLR4 were observed nearby the HBsAg-expressing hepatocytes. The HBV antigenemia as well as the replication and transcription were higher in TLR4-mutant C3H/HeJ mice than in normal C3H/ HeN mice. The HBV-specific immune responses were impaired in the liver or spleen of the C3H/HeJ mice. Their inducible nitric oxide synthase （iNOS） expression on the hepatic infiltrating cells was also impaired. When adoptively transferring splenocytes from C3H/HeN mice to C3H/HeJ mice, the HBV replication was inhibited to the level as that of C3H/HeN. CONCLUSION： These results suggest that TLR4 plays an anti-HBV role in vivo through the induction of iNOSexpression and HBV-specific immune responses after HBV expression.

Possible mechanism for hepatitis B virus X gene to induce apoptosis of hepatocytes

AIM： To investigate the possible mechanism for HBV X gene to induce apoptosis of hepatocyte HL-7702 cells.METHODS： HBV X gene eukaryon expression vector pcDNA3-X was established and transfected into HL-7702 cells by lipid-mediated transfection, including transient and stable transfection. Positive clones were screened by incubating in the selective medium with 600 μg/mL G418 and named HL-7702/HBV-encoded X protein （HBx） cells. The expressions of Fas/FasL, Bax/Bcl-2, and c-myc mRNA were measured by semi-quantitative RT-PCR in HL-7702/HBx and control group, respectively.RESULTS： RT-PCR analysis confirmed that HBV X gene was transfected into HL-7702 cells successfully. By semiquantitative RT-PCR analysis, Bax and c-myc mRNA levels in HL-7702/HBx cells of transient transfection were significantly higher than those in control, FasL and c-myc mRNA levels in HL-7702/HBx cells of stable transfection were significantly higher than those in control, whereas the Bcl-2 mRNA levels in HL-7702/HBx cells of transient and stable transfection were significantly lower than thosein control.CONCLUSION： HBV X gene may promote the apoptosis of hepatocytes by regulating the expressions of Fas/FasL, Bax/Bcl-2, and c-myc gene in a dose-dependent manner.

Hepatitis B viral infection in maintenance hemodialysis patients:A three year follow-up

AIM： To observe the prevalence of hepatitis B virus （HBV） infection in maintenance hemodialysis patients. METHODS： Eighty-eight hemodialysis patients who had been receiving hemodialysis regularly for an average of 39.45 ± 7.57 （range from 36 to 49） rno were enrolled in this study. HBV markers were measured in these patients before hemodialysis and in 100 healthy controls by the chemiluminescent microparticle immunoassay （CMI） method in order to compare the incidence of HBV infection in hemodialysis patients versus normal healthy people. All patients were then divided into two groups： patients positive for HBV markers （i.e. those positive for HBsAg, anti-HBc, HBeAg, anti-HBe, with or without positive anti-HBs） （n = 33）, and patients negative for HBV markers （including those only positive anti-HBs） （n = 55）. The following information was obtained for all patients： socio-demographic data, number of blood transfusions and some laboratory investigations. After 39.45 ± 7.57 mo follow-up, HBV markers were measured in these patients by CMI. RESULTS： The incidence of HBV infection in maintenance hemodialysis patients was 37.5%, which was higher than in controls （9%）. In the patients positive for HBV markers, there were 13 patients （39.4%） who had a history of blood transfusion, which was more than the number [12 （21.8%）, P = 0.04] of patients negative for HBV markers. Eight of the 88 patients negative for HBV markers turned out to be positive, while three of the 33 patients positive for HBV markers turned out to be negative. There was no cirrhosis of the liver or hepatoma occurring in these patients. CONCLUSION： Maintenance hemodialysis patientshave a higher risk of HBV infection than the average population. The number of blood transfusions is associated with an increased prevalence of HBV. While it is hard for hemodialysis patients to eliminate HBV, the prognosis of patients with positive HBV markers is good.

Significance of hepatitis B virus surface antigen, hepatitis C virus expression in hepatocellular carcinoma and pericarcinomatous tissues

AIM： To investigate the correlation between hepatitis B virus surface antigen （HBsAg）, hepatitis C virus （HCV） expression in hepatocellular carcinoma （HCC）, the HAI score of the noncancerous region of the liver and the serum Alpha fetoprotein （AFP） level. METHODS： The patterns of HBsAg and HCV in 100 cases of HCC and their surrounding liver tissues were studied on paraffin-embedded sections with immunohistochemistry, the histological status was determined by one pathologist and one surgeon simultaneously using the hepatitis activity index （HAIl score, and AFP was detected by radioimmunity. The study included 100 consecutive patients who underwent curative resection for HCC. Based on HBsAg and HCV expression, the patients were classified into 4 groups： patients positive for HBsAg （HBsAg group）, patients positive for HCV （HCV group）, patients negative for both HCV and HBsAg （NBNC group） and patients positive for both HBsAg and HCV （BC group）. RESULTS： The BC group had significantly higher HAI scores than the other three groups. （BC 〉 HCV 〉 HBsAg 〉 NBNC）. HBV and HCV virus infection was positively correlated with HAI （rs = 0.39, P = 0.00011. The positive rate of AFP （85.7%） and the value of AFP （541.2 ng/mL） in the group with HBV and HCV co-infection were the highest among the four groups. The positive rate （53.3%） of AFP and the value of AFP （ 53.3 ng/mL） in the group with none-infection of HBV and HCV were the lowest. HBV and HCV virus infection was positively correlated with AFP（rs = 0.38, P = 0.0001）. CONCLUSION： The AFP increase in patients with liver cancer was positively correlated with the infection of HBV and HCV. The-serum AFP elevation by the infection of HBV and HCV is one of mechanisms which lead to hepatocarcinogenesis, and the antivirus intervening treatment of hepatitis is significant for the prognosis of liver cancer. From our Spearman＇s rank correlation analysis, we can conclude that the severity of virally induced inflammation is correlated with HBsAg and HCV expression in HCC tissues and noncancerous tissues. Prior co-infection of HBV in HCV patients may be an adverse risk factor for intrahepatic inflammation.

Study of transactivating effect of pre-S2 protein of hepatitis B virus and cloning of genes transactivated by pre-S2 protein with suppression subtractive hybridization

AIM： To investigate the transactivating effect of pre-S2 protein of hepatitis B virus （HBV） and construct a subtractive cDNA library of genes transactivated by pre-S2 protein with suppression subtractive hybridization （SSH） technique, and to pave the way for elucidating the pathogenesis of HBV infection. METHODS： pcDNA3.1（-）-pre-S2 containing pre-S2 region of HBV genome was constructed by routine molecular methods. HepG2 cells were cotransfected with pcDNA3.1 （-）-pre-S21pSV-lacZ and empty pcDNA3.1（-）/pSV-lacZ. After 48 h, cells were collected and detected for the expression of β-galactosidase （β-gal）. SSH and bioinformatics techniques were used, the mRNA of HepG2 cells transfected with pcDNA3.1（-）-pre-S2 and pcDNA3.1（-） empty vector was isolated, respectively, cDNA was synthesized. After digestion with restriction enzyme RsaI, cDNA fragments were obtained. Tester cDNA was then divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR, amplified cDNA fragments were subcloned into pGEM-Teasy vectors to set up the subtractive library. Amplification of the library was carried out with E.coli strain DH5α The cDNA was sequenced and analyzed in GenBank with Blast search after PCR. RESULTS： The pre-S2 mRNA could be detected in HepG2 cells transfected with pcDNA3.1（-）-pre-S2 plasmid. The activity of β-gal in HepG2 cells transfected with pcDNA3.1 （-）-pre-S2/pSV-lacZ was 7.0 times higher than that of control plasmid （P〈0.01）. The subtractive library of genes transactivated by HBV pre-S2 protein was constructed successfully. The amplified library contains 96 positiveclones. Colony PCR showed that 86 clones contained 200-1 000 bp inserts. Sequence analysis was performed in 50 clones randomly, and the full length sequences were obtained with bioinformatics method and searched for homologous DNA sequence from GenBank, altogether 25 coding sequences were obtained, these cDNA sequences might be the target genes transactivated by pre-S2 protein. CONCLUSION： The pre-S2 protein of HBV has transactivating effect on SV40 early promoter. The obtained sequences may be target genes transactivated by pre-S2 protein among which some genes coding proteins involved in cell cycle regulation, metabolism, immunity, signal transcluction and cell apoptosis.This finding brings some new clues for studying the biological functions of pre-S2 protein and further understanding of HBV hepatocarcinogesis.

Novel Evidence Suggests Hepatitis B Virus Surface Proteins Participate in Regulation of HBV Genome Replication

Naturally occurring mutations in surface proteins of Hepatitis B virus(HBV) usually result in altered hepatitis B surface antigen(HBsAg) secretion efficiency.In the present study,we reported two conserved residues,M75 and M103 with respect to HBsAg,mutations of which not only attenuated HBsAg secretion(M75 only),but also suppressed HBV genome replication without compromising the overlapping p-gene product.We also found M75 and M103 can initiate truncated surface protein(TSPs) synthesis upon over-expression of full-length surface proteins,which may possibly contribute to HBV genome replication.However,attempts to rescue replicationdefective HBV mutant by co-expression of TSPs initiated from M75 or M103 were unsuccessful,which indicated surface proteins rather than the putative TSPs were involved in regulation of HBV genome replication.

Expression of heat shock proteins (HSP27, HSP60, HSP70, HSP90, GRP78, GRP94) in hepatitis B virus-related hepatocellular carcinomas and dysplastic nodules

AIM: Expression of heat shock proteins (HSPs) is frequently up-regulated in hepatocellular carcinoma (HCC), which evolves from dysplastic nodule (DN) and early HCC to advanced HCC. However, little is known about the differential expression of HSPs in multistep hepatocarcinogenesis. It was the purpose of this study to monitor the expression of HSPs in multistep hepatocarcinogenesis and to evaluate their prognostic significance in hepatitis B virus (HBV)related HCC.METHODS: Thirty-eight HCC and 19 DN samples were obtained from 52 hepatitis B surface antigen-positive Korean patients. Immunohistochemical and dot immunoblot analyses of HSP27, HSP60, HSP70, HSP90, glucoseregulated protein (GRP)78, and GRP94 were performed and their expression at different stages of HCC development was statistically analyzed.RESULTS: Expression of HSP27, HSP70, HSP90, GRP78, and GRP94 increased along with the stepwise progression of hepatocarcinogenesis. Strong correlation was found only in GRP78 (Spearman's r= 0.802). There was a positive correlation between the expressions of GRP78, GRP94, HSP90, or HSP70 and prognostic factors of HCC. Specifically, the expression of GRP78, GRP94, or HSP90 was associated significantly with vascular invasion and intrahepatic metastasis.CONCLUSION: The expressions of HSPs are commonly up-regulated in HBV-related HCCs and GRP78 might play an important role in the stepwise progression of HBVrelated hepatocarcinogenesis. GRP78, GRP94, and HSP90 may be important prognostic markers of HBV-related HCC, strongly suggesting vascular invasion and intrahepatic metastasis.

Positive Rate of Different Hepatitis B Virus Serological Markers in Peking Union Medical College Hospital,a General Tertiary Hospital in Beijing

Objectives To investigate the positive rate of different hepatitis B virus （HBV） serological markers, and the demographic factors related to HBV infection. Methods We enrolled all patients tested for HBV serological markers, such as HBV surface antigen （HBsAg）, HBV surface antibody （HBsAb）, hepatitis B e antigen （HBeAg）, hepatitis B e antibody （HBeAb）, HBV core antibody （HBcAb）, and HBV-DNA from July 2008 to July 2009 in Peking Union Medical College Hospital. The positive rate of each HBV serological marker was calculated according to gender, age, and department, respectively. The positive rates of HBV-DNA among patients with positive HBsAg were also analyzed. Results Among 27 409 samples included, 2681 （9.8%） were HBsAg positive. When patients were divided into 9 age groups, the age-specific positive rate of HBsAg was 1.2%, 9.6%, 12.3%, 10.9%, 10.3%, 9.7%, 8.0%, 5.8%, and 4.3%, respectively. The positive rate of HBsAg in non-surgical department, surgical department, and health examination center was 16.2%, 5.8%, and 4.7%, respectively. The positive rate of HBsAg of males （13.3%） was higher than that of females （7.3%, P=0.000）. Among the 2681 HBsAg （＋） patients, 1230 （45.9%） had HBV-DNA test, of whom 564 （45.9%） were positive. Patients with HBsAg （＋）, HBeAg （＋）, and HBcAg （＋） result usually had high positive rate of HBV-DNA results （71.8%, P=0.000）. Conclusions Among this group of patients in our hospital, the positive rate of HBsAg was relatively high. Age group of 20-29, males, and patients in non-surgical departments were factors associated with high positive rate of HBsAg.

Relationship between body surface area and ALT normalization after long-term lamivudine treatment

AIM： To further evaluate the relationship between BSA and the effects of lamivudine in a greater number of cases and over a longer period of observation than in our previous evaluation. METHODS： We evaluated 249 patients with chronic hepatitis B. The effects of treatment for one year （n = 249）, two years （n = 147）, and three years （n = 72） were evaluated from the levels of serum ALT and HBVoDNA, as biological and virological effects （undetectable levels by PCR）, respectively. Moreover, several variables that could influence the response to treatment, including ALT, albumin, bilirubin, platelet counts, BSA, HBVoDNA, and HBeAg were analyzed. RESULTS： For 1-year treatment, multivariate analysis revealed that BSA （P = 0.0002） was the only factor for the biological effect, and that ALT （P = 0.0017）, HBV- DNA （P = 0.0004）, and HBeAg （P = 0.0021） were independent factors for the virological effect. For 2-year treatment, multivariate analysis again showed that BSA （P = 0.0147） was the only factor for the biological effect, and that ALT （P = 0.0192） and HBeAg （P = 0.0428） were independent factors for the virological effect. For 3-year treatment, multivariate analysis, however, could not reveal BSA （P = 0.0730） as a factor for the normalization of ALT levels. CONCLUSION： BSA is a significant predictor for the normalizing the effect of lamivudine therapy on ALT for an initial 2-year period, suggesting that lamivudine dosage should be based on the individual BSA.

Effects of hepatitis B virus on p53 expression in hepatoma cell line SMMU-7721

AIM： To investigate the contribution of HBV in the development of hepatocarcinoma by examining the effects of HBV on p53 function in SMMU-7721 cell line, METHODS： Ptasrnid pCMVp53 was transfected or cotransfected with pCMVHBVa （wild-type HBV） or PCMVHBVb （mutation type HBV） into the hepatoma cell line SMMU-7721 by lipofectamine. Apoptosis cells were labeled with annexin V-FITC and confirmed by flow cytometry. Reporter plasmid PG13-CAT or p21-1uc was cotransfected, respectively, into each group to determine the transactivation activity of p53 and its effect on p21 promoter. Western blot was performed to observe p53 expression in hepatoma cell line of each group. RESULTS： The group transfected with pCMVp53 alone exhibited higher luciferase activity and higher apoptosis rate, otherwise, the p53 expression and reporter activity of PG13-CAT or P21-1uc as well as cell apoptosis rate were obviously higher in the group cotransfected of pCMVp53 with pCMVHBVa, but not in the other cotransfected group, CONCLUSION： Transient transfection of HBV into the SMMU-7721 cell line can enhance p53 expression and its effects on development of hepatocarcinoma.

Transformation of hepatitis B serologic markers in babies born to hepatitis B surface antigen positive mothers

AIM:To better understand the clinical significance of hepatitis B seroiogic markers in babies born to hepatitis B surface antigen (HBsAg) positive mothers, the incidence of maternal seroiogic markers of hepatitis B via placenta and its transformation in these babies were investigated. METHODS: Mothers with positive HBsAg were selected in the third trimester of pregnancy. Their babies received immunoprophylaxis with hepatitis B immunoglobulin and hepatitis B vaccine after birth, and were consecutively followed up for hepatitis B seroiogic markers and HBV DNA at birth, mo 1, 4, 7, 12, and 24. RESULTS: Forty-two babies entered the study, including 16 born to hepatitis B e antigen (HBeAg)-positive HBsAg carrier mothers and 26 to HBeAg-negative HBsAg carrier mothers. Apart from four babies born to HBeAg-positive carrier mothers and demonstrated persistent positive HBeAg eventually became HBV carriers, all other babies developed anti-HBs before 12 mo of age. Among the other 12 babies born to HBeAg-positive carrier mothers, HBeAg was detected in 7 at birth, in 4 at mo 1, and in none of them thereafter. No antibody response to the transplacental HBeAg was detected. Among the babies born to HBeAg-negative carrier mothers, anti-HBe was detected 100% at birth and mo 1, in 88.5% at mo 4, in 46.2% at mo 7, in 4.2% at mo 12 and none in mo 24. Among all the immunoprophylaxis-protected babies born to either HBeAg-positive or HBeAg-negative carrier mothers, anti-HBc was detected in 100% at birth, mo 1 and mo 4, in 78.9% at mo 7, in 36.1% at mo 12 and in none at mo 24. CONCLUSION: HBeAg can pass through human placenta from mother to fetus and become undetectable before 4 mo of age, but no antibodies response to the transplacental HBeAg can be detected till mo 24 in the immunoprophylaxis-protected babies. The sole existence of anti-HBe before 1 year of age or anti-HBc before 2 years of age in babies born to HBsAg carrier mothers may simply represent the transplacental maternal antibodies, instead of indicators of HBV infection status.

Character of HBV (hepatitis B virus) polymerase gene rtM204V/I and rtL180M mutation in patients with lamivudine resistance

Objectives: To investigate the relationship between HBV (hepatitis B virus) polymerase gene 180 and 204 sites mutation and lamivudine resistance. Methods: One hundred forty-one patients with lamivudine resistance after lamivudine treatment and 60 chronic hepatitis B patients without lamivudine treatment were enrolled in this study. The serum HBV DNA mutation was analyzed by sequence detection via polymerase chain reaction (PCR). The sequences of the same patient were analyzed before and after lamivudine treatment. Results: One hundred and nine lamivudine resistance patients had HBV YMDD (tyrosine-methionine-aspartate-aspartate) mutation. Among them, 45 patients had rtL 180M/M204V mutation (41.28%), 28patients had rtL180M/M204I mutation (25.70%) and 36 patients had rtM204I mutation (33.02%). There were 6 patients with rtL180M mutation in 32 lamivudine resistance patients. Sixty chronic hepatitis patients without lamivudine treatment had no mutations. Conclusions: HBV mutations, which play an important role in lamivudine resistance usually locate at polymerase gene 204 site; 180 site mutation was also observed in these patients. Evaluation of the anti-virus therapy by surveillance of the two sites mutations is of importance.

A prophylactic approach for bone marrow transplantation from a hepatitis B surface antigen-positive donor

It has been accepted that bone marrow transplantation （BMT） is the only curative therapeutic option for certain hematologic malignancies. The southeast Asia region is an endemic area of hepatitis B virus （HBV） infection; thus, BMT using a hepatitis B surface antigen （HBsAg）- positive donor is occasionally unavoidable. Organ transplantation using a HBsAg-positive donor can lead to post-transplantation de novo HBV infection and severe HBV-related hepatitis if no effective prophylactic measures are taken prior to and after transplantation. In this report, a four-level approach was designed for a patient with chronic myeloid leukemia, beginning with a booster HBV vaccination before performing BMT with a HBsAg-positive donor. Prior to BMT, the HBV viral load of the donor was reduced to an undetectable level by anUviral therapy. After BMT, hepatitis B immunoglobulin was administered intramuscularly for 1 wk together with a long-term antiviral drug, lamivudine. One year after discontinuation of lamivudine, the patient is still free of HBV infection.

Gene expression analysis of primary normal human hepatocytes infected with human hepatitis B virus

AIM： To find the relationship between hepatitis B virus （HBV） and hepatocytes during the initial state of infection by cDNA microarray. METHODS： Primary normal human hepatocytes （PNHHs） were isolated and infected with HBV. From the PNHHs, RNA was isolated and inverted into complement DNA （cDNA） with Cy3- or Cy5- labeled dUTP for microarray analysis. The labeled cDNA was hybridized with microarray chip, including 4224 cDNAs. From the image of the microarray, expression profiles were produced and some of them were confirmed by RT-PCR, immunoblot analysis, and NF-κB luciferase reporter assay. RESULTS： From the cDNA microarray, we obtained 98 differentially regulated genes. Of the 98 genes, 53 were up regulated and 45 down regulated. Interestingly, in the up regulated genes, we found the TNF signaling pathway-related genes： LT-α, TRAF2, and NIK. By using RT-PCR, we confirmed the up-regulation of these genes in HepG2, HuhT, and Chang liver cells, which were transfected with pHBV1.2x, a plasmid encoding all HBV messages. Moreover, these three genes participated in HBV- mediated NF-κB activation. CONCLUSION： During the initial state of HBV infection, hepatocytes facilitate the activation of NF-κB through up regulation of LT-α, TRAF2, and NIK.