Objective To construct recombinant full length genotype B and C hepatitis B virus (HBV)and to examine HBV DNA replication and hepatitis B surface antigen(HBsAg),hepatitis B e antigen(HBeAg)expressions in Huh7 cells. Methods The full length genotype B and C HBV DNA were extracted and amplified from two HBV infected patients. The recombinant plasmids were constructed by inserting the amplified HBV fragments into the eukaryotic expression vector,pHY106,which were then transfected into Huh7 cells. The cells transfected with blank pHY106 vector were used as control. HBV DNA replication at 72 hours of transfection was detected by Southern blot. The HBV DNA levels in Huh7 cells at 24,48,72,96 and 120 hours of transfection were determined by real-time polymerase chain reaction(PCR).Meanwhile, the HBsAg and HBeAg expression levels in the supernatants at 24,48,72,96 and 120 hours were determined by enzyme linked immunosorbent assay(ELISA).Results The recombinant plasmids expressing genotype B or C HBV DNA were successfully constructed.The HBV replicative intermediates in HBV core particles,including rcDNA dsDNA and ssDNA,were detected by Southern blot.HBV DNA level could reach 8 lg copy/mL which was by real-time PCR. HBsAg and HBeAg levels determined by ELISA peaked at 72 hours after transfection and then declined gradually. Conclusions The recombinant plasmids inserted with genotype B or C HBV DNA are constructed successfully, which can express high levels of HBsAg and HBeAg in Huh7 cells. This system provides a platform for studying the pathogenesis of B and C genotype HBV, the interaction between HBV and host, as well as exploiting new drugs against HBV.

Streptomyces sp. A048 was cultured in a complete medium to the last stage of log phase,the hyphae were washed and collected by centrifugation. Then the hyphae were inoculated in liquid medium for chitinase production using two-step fermentation. Activity of chitinase produced by two-step fermentation was 1.1 times higher than that from one-step fermentation,and ferment cycle was for 54 hours,which was 66 hours shorter than that of one-step fermentation. The hyphae and the powder of chitin were co-immobilizated and cultured in liquid medium for 36 hours,activity of chitinase was 1.8 times higher than that from one-step fermentation,and ferment cycle was 54h shorter than that of one-step fermentation. By adding 0.4% cellulose to two-step fermentation,activity of chitinase was 18.52 U/mL that was 4 times higher than that from the control and 10 times higher than that from one-step fermentation. Two step fermentation with chitin and cellulose may be the optimal fermentation process to produce Chitinase from Streptomyces sp. A048.

To investigate the effects of rennin angiotensin system blockade on the microvessel density in islets of diabetic rats and its relationship with islet function, diabetes model was created by feeding of high-caloric laboratory chow plus intraperitoneal injection of a small dose of streptozotocin (30 mg/kg). After 8 weeks intervention with perindopril (AE, n=10) or valsartan (AR, n=10), the islet function of the animals was evaluated by intravenous insulin release test (IVIRT). The pancreases were immunohistochemically stained to analyze the content of insulin and vascular endothelial growth factor (VEGF) in the islets. The microvessel density (MVD) of islets was detected by counting CD34 positive cells. The hypoxia inducible factor (HIF)-1alpha mRNA expression in the islets was detected by RT-PCR. Compared with normal control group (NC, n=10), the area under the curve for insulin from 0 to 30 min (AUCI(0-30)) of diabetes group (DM, n=8) was decreased by 66.3%; the insulin relative concentration (IRC) of betacell was decreased significantly; the relative content of VEGF was increased obviously [(-4.21+/-0.13) vs (-4.06+/-0.29)]; MVD in islets was decreased by 71.4%; the relative expression of HIF-1alpha mRNA was increased by 1.19 times (all P<0.01). Compared with DM group, the AUCI(0-30) of AE and AR group was increased by 44.6% and 34.9% respectively; IRC was also increased significantly; the relative content of VEGF was decreased by 21.2% and 21.7% respectively; MVD was increased by 62.5% and 75.0% respectively; the relative expression of HIF-1alpha was decreased by 27.2% and 29.0% respectively (all P<0.01 or P<0.05). There were no significant differences in the said indexes between group AE and AR. It is concluded that the blockade of RAS may ameliorate islets function of diabetic rats by increasing the MVD in islets.

OBJECTIVE: To explore the treatment and possible prognostic factor of nasal sinuses adenoid cystic carcinoma. METHOD: Retrospectively analysed the records of 18 patients with complete clinical and pathological data,which including 4 patients given up treatment, 5 patients taken surgical treatment and 9 patients taken surgical treatment as well as radiotherapy and chemotherapy. RESULT: Fifty percent of the patients got 2-year survival and 3 cases of death due to intracranial tumor invasion and 2 patients died of the disease distant metastasis. CONCLUSION If patients got Nasal sinuses adenoid cystic carcinoma, they should take comprehensive treatment based on surgery, in order to improve the survival rate. The prognosis depends on the tumor early detection and early treatment, the sooner the treatment, the better.
Adult ; Aged ; Carcinoma, Adenoid Cystic ; therapy ; Female ; Humans ; Male ; Middle Aged ; Nasal Cavity ; pathology ; Paranasal Sinus Neoplasms ; therapy ; Prognosis ; Retrospective Studies

The survey of traditional knowledge related to Chinese materia medica resources is the important component and one of the innovative aspects of the fourth national survey of the Chinese materia medica resources. China has rich traditional knowledge of traditional Chinese medicine (TCM) and the comprehensive investigation of TCM traditional knowledge aims to promote conservation and sustainable use of Chinese materia medica resources. Building upon the field work of pilot investigations, this paper introduces the essential procedures and key methods for conducting the survey of traditional knowledge related to Chinese materia medica resources. The essential procedures are as follows. First is the preparation phrase. It is important to review all relevant literature and provide training to the survey teams so that they have clear understanding of the concept of traditional knowledge and master key survey methods. Second is the field investigation phrase. When conducting field investigations, survey teams should identify the traditional knowledge holders by using the 'snowball method', record the traditional knowledge after obtaining prior informed concerned from the traditional knowledge holders. Researchers should fill out the survey forms provided by the Technical Specification of the Fourth National Survey of Chinese Materia Medica Resources. Researchers should pay particular attention to the scope of traditional knowledge and the method of inheriting the knowledge, which are the key information for traditional knowledge holders and potential users to reach mutual agreed terms to achieve benefit sharing. Third is the data compilation and analysis phrase. Researchers should try to compile and edit the TCM traditional knowledge in accordance with intellectual property rights requirements so that the information collected through the national survey can serve as the basic data for the TCM traditional knowledge database. The key methods of the survey include regional division of Chinese materia medica resources, interview of key information holders and standardization of information.' In particular, using "snowball method" can effectively identify traditional knowledge holder in the targeted regions and ensuring traditional knowledge holders receiving prior informed concerned before sharing the information with researcher to make sure the rights of traditional knowledge holders are protected. Employing right survey methods is not only the key to obtain traditional knowledge related to Chinese materia medica resources, but also the pathway to fulfill the objectives of access and benefit sharing stipulated in Convention on Biological Resources. It will promote the legal protection of TCM traditional knowledge and conservation of TCM intangible, cultural heritage.
China ; Data Collection ; Databases, Pharmaceutical ; Drugs, Chinese Herbal ; Materia Medica ; Medicine, Chinese Traditional

Objective To construct prokaryotic expression vector carrying jc virus(JCV)t-antigen gene,express and purify this fusion protein.Methods The JCV t-antigen gene from a cerebrospinal fluid sample was amplified using polymerase chain reaction(PCR)method.After sequencing.the gene was cloned into plasmid pET32a(+)to construct recombinant prokaryotic expression vector pET32a(+)-t.The t-antigen fusion protein was expressed by isopropy-~D-thiogalactoside(IPTG)induction and prepared in large scale,then purified by Ni+affinity column chromatography.The polyclonal antibody was obtained from the BAI.B/C mouse immunity by the purified protein.Results The relative molecular nlass of recombinant protein expressed by pET32a(+)-t was about 41 000.Sodium dodeeylsulfate-polyaerylamide gel electrophoresis(SDS-PAGE)showed that the fusion protein W&S highly expressed after 3.5～20.Oh of IPTG induction.The antigenicity of the purified protein Was well confirmed by Western blot.The anti-mousepolyclonal antibody was obtained successfully from immunized BALB/c mice.Conclusions The prokaryotic expression vector pET32a(+)-t is successful constructed and the fusion protein is expressed and purified.Furthermore,the antibody of JCV small envelop protein t is successfully prepared.This work provides vMuable information for further study on epidemiology and biological function of t antigen.

Objective To express nonstructural protein 2 transactivated protein (NS2TP) of hepatitis C virus (HCV) in the prokaryotic expression system and prepare polyclonal antibody,and to study the expressions in different liver tissues.Methods NS2TP gene was amplified by polymerase chain reaction (PCR) technique and cloned into the prokaryotic expression vector pET-32a(+),which was transformed into E.coli BL21.The protein was induced by isopropyl thiogalactose (IPTG) and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and confirmed by Western blotting assay.The recombinant protein were expressed and purified in a large amount.The rabbit was immunized with the purified protein to prepare polyelonal antibody.The liver tissues of patients with chronic HCV infection and healthy controls were detected by immunohistochemistry method.Results The recombinant NS2TP protein (relative molecular mass:33×103 ) and polyclonal antibody with high titer and specificity were successfully prepared.NS2TP expressions in the liver of patients with chronic HCV infection were higher than those of healthy controls,and were mainly distributed in the nucleus of hepatocytes.Conclusions The NS2TP expression level and intracellular location in liver tissue of patients with chronic HCV infection are understanded,which could bring new clues for further study of the biological function of NS2TP and the pathogenesis of HCV infection.

Rheumatoid arthritis (RA), as a common systemic inflammatory autoimmune disease, affects approximately 1 in 100 individuals. Effective treatment for RA is not yet available because current research does not have a clear understanding of the etiology and pathogenesis of RA. Xinfeng Capsule, a patent Chinese herbal medicine, has been used in the treatment of RA in recent years. Despite its reported clinical efficacy, there are no large-sample, multicenter, randomized trials that support the use of Xinfeng Capsule for RA. Therefore, we designed a randomized, double-blind, multicenter, placebo-controlled trial to assess the efficacy and safety of Xinfeng Capsule in the treatment of RA.

Objective To evaluate the effectiveness of using high-fidelity simulator (HFS) in medical training of cardiopulmonary resuscitation (CPR).Methods The randomized controlled trials (RCTs),quasi-randomized controlled trials (q-RCTs) about comparing HFS with traditional teaching methods in medical training of CPR were searched from Cochrane Library,Pubmed,Web of Knowledge,CNKI,CBM and Wanfang Data.The methodological quality of the included studies was assessed and the valid data were extracted.Meta-analysis was conducted with the Cochrane Collaboration RevMan 5.0.Results Ten q-RCTs and one RCT were included.The quality of the studies were relatively low.Meta-analysis showed that compared with the traditional teaching method,high-fidelity simulation in medical training of CPR had no statistically significant differences in mastering the theoretical knowledge,but had statistically significant differences in mastering the CPR skills.Conclusions HFS in medical training of CPR skills is positive and effective,superior to the traditional teaching methods,but in terms of theoretical knowledge and satisfaction,self-confidence,more researches still need to be further confirmed.

OBJECTIVETo observe the anti-inflammatory effect of total saponins of Panax japonicus on LPS-induced RAW264. 7 macrophages.

METHODThe effect of total saponins of P. japonicus of different concentrations on RAW264. 7 cell viability was determined with the MTT method. The NO kit assay was adopted to detect the NO release of total saponins of P. japonicus to LPS-induced RAW264. 7 cells. The enzyme linked immunosorbent assay (ELISA) was used to detect the secretion of tumor necrosis factor-alpha (TNF-alpha) and interleukin 1-beta (IL-1beta). The reverse transeriptase-polymerase chain reaction (RT-PCR) was used to determine the expression of inducible nitric oxide synthase (iNOS) ,TNF-alpha,IL-1beta. The protein expression of nuclear transcription factor-kappaB p65 (NF-kappaB p65) was tested by Western blot.

RESULTThe safe medication range of total saponins of P. japonicus was less than 80 mg x L(-1). Compared with the LPS model group, total saponins of P. japonicus high, middle and low dose groups (0.1, 1, 10, 40 mg x L(-1)) could significantly reduce the secretion of NO, TNF-alpha, IL-1beta of LPS-induced RAW264. 7 cells, and inhibit the expressions of iNOS, TNF-alpha and IL-1beta mRNA and the protein expression of NF-kappaB p65.

CONCLUSIONThis study preliminarily proves the protective effect of total saponins of P. japonicus on LPS-induced RAW264.7 macrophages. Its action mechanism may be related to NF-kappaB signal pathway.

Animals ; Anti-Inflammatory Agents ; pharmacology ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Inflammation ; drug therapy ; genetics ; immunology ; Interleukin-1beta ; genetics ; immunology ; Lipopolysaccharides ; adverse effects ; Macrophages ; drug effects ; immunology ; Mice ; NF-kappa B ; genetics ; immunology ; Nitric Oxide ; immunology ; Nitric Oxide Synthase Type II ; genetics ; immunology ; Panax ; chemistry ; Protective Agents ; pharmacology ; Saponins ; pharmacology