Nucleoside analogs (NA) are currently the major anti-hepatitis B virus (HBV) agents in clinic. However,long-term use of NA may initiate a state of drug resistance due to HBV mutation. The genotyping and phenotyping are two basic methods for analysis of HBV drug resistance. Genotyping is done mainly by DNA sequencing and reverse hybridization line probe to detect the well-known drug-resistant mutations. Phenotyping is an essential tool used to identify "novel" and complex resistant mutations,and it is based on the comparison of HBV replication capacities of variants and wild-type counterparts by cell-transfer of individual viral genomes followed by quantitation of HBV replication intermediates in the presence of antivirals in different concentrations. Clinical choice of HBV resistance detection assays relies on their sensitivity,specificity and cost-effectiveness. In our recent studies,some novel HBV resistant features have been found,including multidrug-resistant strain with mutations due to the use of both entecavir and adefovir,association of HBV genotype/subgenotype with the resistant mutational patterns,transmission of the resistant HBV to cause acute hepatitis,replication compensation of rt229 variants,etc. These findings are important for better understanding the clinical features of HBV drug-resistant mutations and strengthening the management of HBV drug resistance in clinic.

Hepatitis B virus(HBV) is a double-strand hepadnavirus.HBV genomics have unique properties:(1) Viral polymerase/reverse transcriptase(RT) is lacking in proofreading activity,leading to higher mutation occurrence than other DNA viruses.(2) Overlapping open reading frames in viral genomics may produce the possibility of mutation of two viral proteins mutation as a result of one-site genetic change.(3) A stable covalently closed circular DNA(cccDNA) form exists as an important intermediate in the life cycle.(4) Viral gene fragments may be integrated into host cellular genomic DNA.(5) Viral proteins could be a trans-regulator for certain host cellular protein expression.The virological characteristics of HBV are closely related with the chronicity and disease course of hepatitis B.Multi-site detection of HBV RT-gene mutations is very important for monitoring drug resistance and rational administration of anti-HBV therapy in clinic.Analysis of viral genomic variation and detection of cccDNA are highly valuable in better understanding virological pathogenesis and evaluate therapeutic efficacy.Methodological innovation and improvement is the basis for the study of virological characteristics of HBV.

Objective To compare the efficiency of detecting YMDD mutations by direct DNA sequencing and real time fluerescence PCR, and to explore the significance of the mutations of drug-resistance gene other than YMDD mutation. Methods 92 serum samples from 89 patients with chronic hepatitis B were collected and all the samples were detected by real-time PCR for YMDD mutation. HBV DNA was extracted from serum and was amplified by nest PCR to achieve HBV P gene RT region sequence. The PCR products were sequenced at both directions, and the sequencing results were contrasted through NTI program. The other 11 known drug-resistance mutation sites at the HBV RT region were also analyzed. Results Among the 37 samples with no YMDD mutation detected by real-time PCR, 33 samples were with M204M (without mutation), 1 sample with M204I and 3 samples with M204V by direct sequencing. Mutation and wild-type standard sequences were all coexisted in the 4 positive samples. There were 7 samples detected with ADV resistant mutation, accounting for 18.9% (7/37). Among the 55 samples with YMDD mutation detected by real-time PCR, 52 samples were detected by DNA sequencing, the accordance rate was 94.5% (52/55); 5 samples with ADV or ETV resistant mutation were detected, accounting for 9.1% (5/55). Conclusions Direct DNA sequencing is a high sensitive, repeatable method to detect drug-resistance mutation at RT region of HBV P gene. The result is well consistent with that attained by real-time PCR. Direct DNA sequencing can also detect various drug-resistance mutations as well as YMDD mutation, which is helpful to generally understand the nucleoside analogue resistant mutation and adopt more reasonable therapy projects against HBV.

Objective To construct the yeast expression vector of a new gene transactivated by hepatitis B virus X protein (XTP11), and to explore the feasibility of cloning the hepatocyte proteins interacting with XTP11 protein by yeast two hybrid system. Methods The XTP11 gene was amplified by polymerase chain reaction (PCR) with specific primers, and the amplified fragment was subcloned into the Nco I/BamH I sites (5′ ends) of yeast expression vector pGBKT7,which was named as pGBKT7(-)-XTP11 encoding fusion proteins of full length of XTP11 and DNA binding domain of yeast protein GAL4. The pGBKT7 (-)-XTP11 plasmid was transformed in the yeast cells AH109 and the expression of XTP11 protein in yeast cells was detected by Western blotting assay. Then the yeast cells were plated on synthetic dropout nutrient medium (SD/-Trp and SD/-Trp-His-Ade) containing X-?-gal, and their autonomous activation was tested. Results The yeast expression vector of XTP11 gene was constructed and the fusion protein in yeast cells was examined. The yeast cells transformed with pGBKT7-XTP11 plasmid could grew well on both of the media, and turned blue on medium containing X-?-gal for the production X-?-galactosidase. This phenomenon suggested that XTP11 protein acted to substitute the functional yeast protein GAL4 activation domain (AD) to activate transcription of reporter genes (ADE2, HIS3, MEL1 and LacZ). Conclusion The XTP11 protein fused to the GAL4 DNA-binding domain functioned as a transcriptional activation domain in yeast, and the transcriptional activation function of XTP11 protein in yeast might restrain researchers to use yeast two hybrid system to clone hepatocyte proteins interacting with XTP11 protein.

Hepatitis B virus (HBV)covalently closed circular DNA (cccDNA)is an intermediate replicative episome which is stable and not easy to eliminate.cccDNA serves as the template for HBV replication and plays an important role in persistence of HBV infection in hep-atocytes,and it is also a biomarker of HBV activity.Studies suggested that HBV has extrahepatic infectivity and cccDNA could be detected in peripheral blood mononuclear cells (PBMC).The predictive values of HBV cccDNA in PBMC for HBV recurrence after liver transplanta-tion and antiviral therapy and mother-to -child transmission of HBV in patients with hepatitis B,as well as the possible mechanism of cccDNA involved in extrahepatic HBV infection,are summarized.Therefore,the detection of HBV cccDNA is of great significance for the study and treatment of HBV.

Objective　To evaluate the clinical efficiency of diode laser transscleral cyclophotocoagulation(TSCPC) for the treatment of refractory glaucoma.Methods　TSCPC were performed in 23 eyes of 23 patients with medically and surgically uncontrolled glaucoma. The laser was operated with a power of 2～3W,duration of 1.5～2s and laser spots(18) were evenly placed 270 around the limbus except temporal. Followed for 3～24 months (average:11 months) . Results　The mean intraocular pressure before and after treatment in 23 eyes were 6.10kPa±2.34kPa and 2.55kPa±1.22kPa respectively,which was statistically significant (P<0.01). IOP below 2.80kPa was obtained in 20 eyes (86.9%). At the time of last visit,the post-operative visual acuity was partly improved besides NLP.Conclusion　Diode laser transscleral cyclophotocoagulation is effective in treating refractory glaucoma. Its advantages are low ratio of ocular hypotension,no atrophy of eyeball,no pain, acceptability,reoperation and therapeuty in clinic and so on.

Objective To analyze the variation in CTL epitope of hepatitis B virus (HBV) popular in China. Methods Amino acid sequences of 13 cytotoxic T lymphocyte (CTL) epitopes previously reported abroad were taken as reference sequences, and comparison was made draw between these reference sequences and both the 45 CTL-epitopic sequences obtained from the Genbank and also 5 CTL-epitopic sequences from native patients with chronic hepatitis B, using Vector NTI software. Results Sequence variations were observed frequently within several CTL eptitopes of HBV genotype B and C compared with the reference sequences. In the 13 analyzed CTL epitopes, 4 showed variation accounting over 70% in genotype B, and 2 have variance over 70% in occurrence in genotype C. Among these, C18-27 (FLPSDFFPSV), HBcAg-derived CTL epitope which was the most frequently studied previously, is showed in variation more frequently in HBV genotype B and C popular in China, reaching 71% and 97%, respectively. Conlusion Some CTL epitopic sequences of HBV genotype B and C popular in China have their own charactiristic difference from previously reported ones, which should be considered in the study of CTL response of Chinese subjects.

Objective To screen the down-regulated genes in HepG2 cells transfected by pcDNA3.1(-)-IFN-? to further investigate the biological mechanism of IFN-?. Methods pcDNA3.1(-)-IFN-? was transfected into HepG2 cells as treatment group and the pcDNA3.1(-) transfected cells as the control to perform suppression subtractive hybridization (SSH). The subtractive library for down-regulated genes was obtained when pcDNA3.1(-) transfected HepG2 as tester and pcDNA3.1(-)-IFN-? transfected cells as driver. A housekeeping gene, G3PDH, was used to estimate the subtractive efficiency. Genes with lower expressions in IFN-? transfected HepG2 cells were obtained from the library after being randomly sequenced and analyzed. Among the obtained genes, regulation of IFN-? on heat shock 90kD (Hsp90) mRNA was further investigated by RT-PCR. Results G3PDH was subtracted efficiently indicating that the subtractive library was constructed successfully. 50 positive clones were randomly isolated for PCR identification. Results showed that most of the plasmids in the clones contained 200-1 000bp inserts. The down-regulated genes obtained were as follows: eukaryotic translation elongation factor 1 beta, ferritin, RAD23 homolog B, signal sequence receptor 2 (SSR2), tissue factor pathway inhibitor, heat shock 90kD protein 1, and adenosylmethionine decarboxylase 1. RT-PCR showed that hsp90 mRNA had a reduced expression in pcDNA3.1 (-)-IFN-? transfected HepG2 cells. Conclusion The down-regulated cDNA subtractive library in HepG2 cells after pcDNA3.1 (-)-IFN-? transfection was constructed successfully. IFN-? could down-regulate the hsp90 mRNA expression.

Objective To investigate the expression of ERBB3 protein,which encoded by ERBB3 gene,in gastric cancer(intestinal type and diffuse type)as well as the normal tissue,and to elucidate the possible role of ERBB3 gene in the progress of gastric cancer.Methods 65 samples of gastric cancer tissue(of which 43 samples were intestinal type and 22 were diffuse type,classified according to the Lauren patho-classification)and 65 samples of normal tissue(control group)were investigated immunohistochemically.The expression of ERBB3 in both control group and gastric cancer group was determined.Comparisons were made according to different pathological types and TNM clinical stages within and between groups.Results 7 of 65 samples(10.8%)in gastric cancer group were found to over-express the ERBB3 protein,while only 1 of 65 samples(1.6%)in control group was found to over-express the ERBB3 protein.Statistical differences(P

To compare the clinical effect and side-effect of intravenous PCA(PCIA) with epidural PCA (PCEA). Method: One hundred and eighty postoperative patients, who were randomly divided into three groups: group PCIA, group PCEA and control, were observed for 3 days after operation. Result: Overall patients in two PCA groups were satisfied with the postoperative analgesia. The incidence of urinary retention in group PCIA were significantly lower than that in group PCEA (P0.05). 10% in group PCIA were in medium sedation whereas no eases in group PCEA were in sedation. At 6th hour after operation,serum cortisol level of control patients was much higher than that of PCA patients. Conclusion=Both PCIA and PCEA have excellent analgesia and reduce stress response after operation. PClA has lower incidence of urinary retention and is performed easily,but inhibits gastrointestinal motility much more and has higher sedative incidence compared with PCEA.