This study was aimed to establish a HPLC method for the content determination of liposoluble components, such as dihydrotanshione I, croptotanshinone, tanshinone I and tanshinone IIA, as well as the content determination of water-soluble components, such as danshensu, protocatechuic aldehyde, rosmarinc acid, salvianolic acid B, and salvianolic acid A in Danshen capsule simultaneously. For liposoluble components, the determination was performed on a Welch ultimate XB-C18 column (250 mm × 4.6 mm, 5 μm) by gradient elution using acetonitrile-water as the mobile phase. The flow rate was 1 mL·min-1. And the detection wavelength was set at 270 nm. For water-soluble components, the determination was performed on a Thermo Syncronis C18 column (250 mm × 4.6 mm, 5μm) by gradient elution using methanol-acetonitrile-0.02%phosphoric acid as the mobile phase. The flow rate was 1 mL·min-1. And the detection wavelength was set at 286 nm. The results showed that there were good linear relationships between components of peak areas and the ranges were 0.472-9.44 μg (r = 0.999 8) for danshensu, 0.352-7.04 μg (r = 0.999 9) for protocatechuic aldehyde, 0.244-4.88 μg (r = 1.000 0) for rosmarinc acid, 2.268-45.36 μg (r = 0.999 9) for salvianolic acid B, 0.168-3.36 μg (r = 0.999 9) for salvianolic acid A, 0.088-1.76 g(r=0.999 9) for dihydrotanshione I, 0.18-3.6μg (r=0.999 9) for croptotanshinone, 0.208-4.16μg (r=0.999 9) for tanshinone I, and 0.17-3.4μg (r=0.999 9) for tanshinone IIA. Average recoveries of the method were between 97.48%and 98.59%. It was concluded that the analysis was stable and reproducible, which can be used as a method for the analysis of Danshen capsule.

Objective To evaluate the importance of clinical screening and follow up by direct colonoscopy for colorectal cancer at an early and curable stage. Methods There were 2 196 elderly people aged between 60 to 89 years. The clinical screening by direct colonoscopy was performed according to the protocol. 1 740 of 2 196(79.2%) patients were followed up every year. Results Fifty two elderly persons were found to be colorectal cancer patients by colonoscopy, with the detectable rate being 2.4%. Nineteen were diagnosed early stage colorectal cancer, accounting for 36.5% of the detected colorectal cancer. Nine among the followed up cases were detected early colorectal cancer, accounting for 45 0% of the detected colorectal cancer. The resectable rate and the 5 year survival rate was 97 7% and 80 9% for colorectal cancer, respectively. 98 9% of the cecum intubation cases was successful. The incidence of complication for colonoscopy was 0 05%. Conclusions By clincal colonscopy screening and follow up study for colorectal cancer and precancerous changes in the elderly, the patients with adenomatoid polyps were early diagnosed and treated, so it raised the detectable rate of early colorectal cancer and the level of grade prevention of colorectal cancer.

Objective To construct a cDNA subtractive library of genes transactivated by c-terminally truncated middle surface protein of hepatitis B virus(MHBs t) with suppression subtractive hybridization technique for cloning genes associated with transactivation. Methods The mRNA was isolated from HepG2 cells transfected with pcDNA3.1(-)-Mt167 and pcDNA3.1(-) empty vectors, respectively, then cDNA was synthesized. After restriction enzyme Rsa I digestion, small-size cDNAs were obtained. Then tester cDNA was 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 and then was subcloned into T/A plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E. coli strain JM109. The cDNA was sequenced and analyzed in GenBank with Blast search after PCR. Results The subtractive library of genes transactivated by MHBs t was constructed successfully. The amplified library contained 94 positive clones. Colony PCR showed that these clones contained 200-800bp inserts. Sequence analysis was performed in 50 clones,and the full length sequences were obtained with bioinformatics method. 23 coding sequences were obtained in total, which consisted of 19 known and 4 unknown ones.Conclusions The obtained sequences may be target genes transactivated by MHBs t, among which some genes coding proteins may involve in cell cycle regulation, immune response and tumour genesis.

Protein-protein binding is the basis of virus and host cell interactions. With the application of technology of studying protein interactions, more knowledge of replication and pathogenesis of hepatitis C virus (HCV) was acquired. Non-structure protein 5B(NS5B) of HCV is a kind of viral protein, which plays an important role in replication of HCV. However, the effect of NS5B is not clear. To investigate the biological function of NS5B, we performed yeast two hybrid to look for proteins in hepatocytes interacting with NS5B. We constructed NS5B bait plasmid by cloning the gene of NS5B into pGBKT7, then transformed it into yeast AH109(a type). The transformed yeast was mated with yeast Y187(? type)containing liver cDNA library plasmid in 2?YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing x-?-gal for screening. Thirty-three colonies were selected and sequenced. Among them, two colonies were new genes with unknown function. The preliminary successful cloning of gene of protein interacting with NS5B paved the way for the study of the physiological function of NS5B and its associated protein.

Objective To screen the HCV NS4A binding protein. Methods By using HCV NS4A as a solidified selective molecule, the T7 select human liver cDNA library was biopanned and the positive clones were selected. After screening, the positive plaques was amplified and then cloned into the pGEM-Teasy vector. Two positive plaques were chosen for DNA sequencing. Results The binding protein of HCV NS4A was identified as mitogen-activated protein kinase (MAPK)-activated protein kinase 5 (MAPKAPK5) by BLAST. Conclusion This approach provides a new way for the study of the pathogenic mechanism of HCV infection.

Objective To screen the HBV core promoter binding protein, and to investigate their potential role in the replication of HBV DNA. Methods By using HBV core promoter being used as a selective molecule, the T7select human liver cDNA library was biopanned and the positive clones were selected. Results After phage display screening, the positive plaques was amplified and then cloned into the pGEM-Teasy vector. Six positive plaques were chosen for DNA sequencing. The binding protein of HBV core promoter was identified as caboxypeptidase N(CPN) by BLAST. Conclusion The results suggest that phage display screening of binding protein of HBV core protein provides a new approach to study the replication mechanism of HBV DNA.

Objective To express soluble human anti-idiotypic single chain Fv to hepatitis C core protein in E.coli. Methods Using phage display technique, the semisynthetic phage library was panned by HCV core monoclonal antibody which was coated in a microtiter plate. After three rounds of biopanning, 53 clones were identified specific to HCV core antibody. The specificity of anti-idiotypic scFv was determined by ELISA. After digested with Sfi/Not, the selected HCV core anti-idiotypic scFv positive clone was subcloned into the vector pCANTAB5E for the expression of E-tagged soluble anti-idiotypic scFv. The E.coli XL1-Blue was transformed and induced by IPTG. The specificity of anti-Id scFv was evaluated with ELISA. Results HCV core anti-Id scFv DNA digestion and sequence data showed that the scFv gene was composed of 774bp. ELISA results demonstrated that the soluble human HCV core anti-idiotypic scFv to HCV core monoclonal antibody had a specific combination character. The molecular weight of expressed HCV core anti-idiotypic scFv was 28kD as shown by SDS-PAGE. Conclusion HCV core anti-Id scFv has been successfully expressed in E.coli.

Objective [WT5”BZ] To study the relationship between human papillomavirus (HPV) 16 and its serum antibody in the development of gastric carcinoma (GC) [WT5”HZ] Methods [WT5”BZ] Polymerase chain reaction (PCR) technique was used to detect HPV DNA in 42 fresh GC specimens and 42 fresh normal gastric mucosa adjacent to the tumour (NGMAT). Enzyme linked immunosorbent assay (ELISA) was used to screen the serum HPV16 antibody(Ab) of 42 GC patients and 46 controls using HPV16 virus like particles (VLPs) which was produced by recombinant bacilovirus in insect cells [WT5”HZ] Results [WT5”BZ] HPV16 DNA was found in 26 2% (11/42) of GC specimens,but in none (0/42) of NGMAT ( P

Objective:To study the effect of Liuwei Dihuang decoction (LW) on secretion of testosterone in the testis leydig cells of senescence accelerated mouse (SAM). Methods:The level of testosterone in the testis leydig cells of SAM with aging and the effect of LW on the secretion of testosterone were observed using cultured testis leydig cells in vitro.Results:The level of testosterone in the testis leydig cells of SAMP8 with aging was significantly decreased and showed a significant difference compared with age-matched SAMR1. Chronic administration of LW (2.5 and 5.0 g/kg) for 5 months significantly ameliorated the secretion of testosterone in SAMP8 compared with control group. Conclusions:The secretory function of testis leydig cells was reduced in SAMP8 with aging. LW ameliorated the secretion of testosterone in the testis leydig cells,indicating that LW could antagonize or delay the deterioration of the testis leydig cells in SAMP8.

Objective To clone a new human gene 5 trans-activated by pre-S1 protein of hepatitis B virus (HBV), PS1TP5, and explore its structure and function by bioinformatics analysis. Methods PS1TP5 was amplified by reverse transcription-polymerase chain reaction (RT-PCR) technique by using HepG2 cDNA as template and inserted into pGEM-T vector by TA cloning. Recombinant eukaryotic expression vector pcDNA TM 3.1/myc-His A-PS1TP5 had been constructed by subcloning, followed by restriction enzyme digestion analysis and sequencing. Bioinformatic methods were used to analyze its possible physical and chemical characters, structure, and function. Results PS1TP5 was successfully amplified and cloned into pGEM-T and pcDNA TM 3.1/myc-His A vector by RT-PCR from HepG2 cDNA. The new gene had been confirmed by sequencing after PCR identification and restriction enzyme digestion and named as PS1TP5 because of its trans-active function. The sequence for the PS1TP5 gene had been deposited into GenBank, the accession number was AY427953. Bioinformatics analysis showed that its ORF was 438bp and translated a protein of 145 aa. Conclusion A new gene-PS1TP5 has been recognized, and its recombinant eukaryotic expression vector (pcDNA TM 3.1/myc-His A-PS1TP5) has been constructed. These results will certainly bring some new clues for the study of the biological function of new gene and pathogenesis of chronic hepatitis B.