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.

To retrospective analyze the clinical profiles of 80 patients undergoing thoracotomy with protection of intercostal nerve versus traditional method.The doses of narcotics of two groups were (12 ± 5)and (43 ± 11) mg respectively.The postoperative levels of visual analogue score (VAS) and such potential complications as pneumonia,atelectasis and paraesthesia were examined (P ＜ 0.01).Protective technique of intercostal nerve during thoracotomy could effectively relieve postoperative chest pain,reduce the dosage of narcotics and lower the occurrence of lung complications.

Objective:To observe effects of Shenkui Decoction on proliferation of ovarian cancer cell.Methods:The Shenkui Decoction- containing rat serum was prepared by using TCM serum pharmacological method,and effects of the drug containing serum on proliferation of ovarian cancer fresh parenchymatous tumor cells were investigated with 3H incorporation method,and effects of the drug-containing serum on proliferation of ovarian cancer cell line Tyk-nu cells were investigated by flow cytometry,cellular activity assay,cellular growth curve assay and cell colony formation rate assay.Results:The drug containing serum could inhibit proliferation of both fresh parenckymatous tumor cells and ovarian cancer cell line Tyk-nu cells,and the inhibitory rate raised with the increase of drug-containing serum content.Conclusion:The Shenkui Decoction-containing rat serum can inhibit proliferation of ovarian cancer fresh parenchymatous tumor cells and ovarian cancer cell line Tyk-nu cells.

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 To study the interaction between HCV core protein and HCBP6 in mammlian cells using CheckMateTM Mammalian Two-Hybrid System.Methods cDNA fragments encoding HCV core protein and HCBP6 were amplified by PCR and subsequently cloned into pGEM-T vector.After verified by sequencing,the target fragments were subcloned into mammalian two-hybrid plasmids,pBIND and PACT,respectively.The recombinant plasmids,pBIND-core and pACT-HCBP6 were co-transfected into HepG2 cells with reporter plasmid pG5luc.pBIND+pACT were induced as background controls,pBIND-Myod+pACT-Id as positive controls,and pBIND-core+pACT,pBIND+pACT-HCBP6 as blank controls.The expression of G5luc,which indicated the interaction between HCV core protein and HCBP6 in mammlian HepG2 cells,was assayed through Dual-Luciferase Report Assay System and Turner Biosystems Veritas Microplate Lunimometer.Results The recombinant vectors pBIND-core and pACT-HCBP6 were successfully constructed.When co-transfected into HepG2 cells with reporter plasmid pG5luc,there were significant differences in the luciferase activity in the pBIND-core and pACT-HCBP6 groups compared with every control group.Conclusions HCBP6 can interact with HCV core protein in HepG2 cells,which provides clues for further study on the function of HCBP6 and core proteins,and on the mechanism of HCV core in cell apoptosis and cancer transformation.

Objective To explore the feasibility and reliability to build a sinoatrial node damage model in canine induced by formaldehyde wet dressing.Methods Twenty dogs were randomly divided into 4 groups(5 in each group) by means of random number table:2-hour,24-hour,1-week and 4-week groups after wet dressing.The sinoatrial node area of canine was damaged by wet dressing with 20% formaldehyde.Sinus node function was measured before,2 h after wet dressing and corresponding times of each group.Electrocardiogram(ECG) of body surface were recorded synchronically.Results Average wet dressing time was 6.2?2.6(3 to 12)min.Five dogs showed significantly decrease of heart rate(HR)(140?11 vs 89?6 beat/min,P