To express a gene of heat shock protein 70(HSP70) in E.coli , HSP70 gene was amplified by PCR. The PCR products were cloned into pUCm T vector and were sequenced; The HSP70 gene was subcloned into vector pET 21a(+) and expressed in E.coli. SDS PAGE and Western blot were employed to identify the expression of the HSP70 gene. Results showed that a fragment about 1 96kb was amplified by PCR. Sequence analysis revealed that the sequence of HSP70 was correct. The HSP70 gene was cloned into pET 21a(+) identified by enzyme digestion and PCR. SDS PAGE and Western blot showed that a M 72 000 protein was expressed and could be recognized by anti HSP70 antibody. Therefore,HSP70 gene has been successfully expressed in E.coli .

Objective To construct a fusion gene vaccine of gastric cancer MG7-Ag mimotope and heat shock protein 70, and to investigate the humoral and cellular immune response in mice induced by the vaccine. Methods The coding sequence of MG7 mimotope was incorporated with HSP70 gene at the 3' terminus by PCR amplification. Then the PCR product was cloned into pcDNA3.1(+) vector to construct the eukaryotic expression vector. The pcDNA3.1/MG7+hsp70 was sequenced to ensure the proper encoding. C57BL/6 mice were immunized with the fusion gene vaccine, and pcDNA3.1/hsp70 and empty pcDNA3.1 were used as controls. The serum was collected at the 3rd week after each immunization. Cellular ELISA was performed to evaluate the induced humoral immunity. The splenocytes were first separated at the 9th week for the assay of antigen specific CTL lysis activity by 51Cr release. Results A fragment about 2.0 kb was obtained by PCR amplification. Sequence analysis revealed that the sequence of mimotope was connected successfully to 3' terminus of hsp70 and the fusion gene was cloned into the pcDNA3.1(+) successfully. Cellular ELISA results suggested that the serum level of MG7-Ag antibody appeared in the vaccinated mice at 9th week, while no MG7-Ag antibody was detected in the controls. The results of the specific lysis rate of splenocytes showed no statistical difference between fusion gene vaccine group and control groups. Conclusion The fusion gene vaccine was constructed successfully and the specific humoral immune response was induced by the fusion gene vaccine.

BACKGROUND: The feasibility and the mechanism of human umbilical cord blood mesenchymal stem cel transplantation for the treatment of liver cirrhosis need to be discussed in-depth. OBJECTIVE: To observe the effect of human umbilical cord blood mesenchymal stem cel transplantation through portal vein on the liver function and tissue pathological changes of the rats with liver cirrhosis. METHODS: Carbon tetrachloride was used to prepare rat model of liver cirrhosis. After the success of modeling, the rats in the cel transplantation group received portal vein injection of 1 mL 5-bromo-2-deoxyuridine -labeled human umbilical cord blood mesenchymal stem cells (5×106), the model group was injected with the same volume of PBS; the normal rats received 1 mL human umbilical cord blood mesenchymal stem cel transplantation via the portal vein were as the control group. At 4 weeks after transplantation, the rat tail vein blood and liver tissue were obtained for testing. RESULTS AND CONCLUSION: At 4 weeks after cel transplantation, compared with the model group, levels of serum alanine aminotransferase, aspartate aminotransferase and total bilirubin in the cel transplantation group were significantly decreased, while the albumin level was increased significantly (P < 0.01); the liver cel inflammatory necrosis, steatosis and liver fibrosis were improved significantly (P < 0.05 or P < 0.01). Immunohistochemistry and immunofluorescence staining showed that human umbilical cord blood mesenchymal stem cel colonization could be seen in the rat liver tissues of the cel transplantation group and control group, but the number of 5-bromo-2-deoxyuridine-positive cells in cel transplantation group was significantly larger than that in the control group. Reverse transcription-PCR test result showed that the expressions of cytokeratin 18 and albumin mRNA could be observed in the rat liver tissue of the cel transplantation group, but no expression could be seen in the control group. It is visible that human umbilical cord mesenchymal stem cells can improve liver function and pathological damage of liver cirrhosis rats in a certain extent, which may relate with the intrahepatic homing colonization and hepatocyte-like cel differentiation of the transplanted cells in the liver cirrhosis rats.

Objective To study the clinical and imaging characteristics of Chinese atopic myelitis (AM) patients.Methods Three diagnosed AM patients were retrospectively analyzed for the clinical data,serum IgE level,antigen specific IgE,cerebrospinal fluid,spinal MRI and therapeutic efficacy profiles.Results All the three patients were male and presented as subacute AM with the onset at 25,47 and 49 years old respectively.Two patients were allergic to pollen and other drugs,while another patient suffered from allergic rhinitis.Elevated serum total IgE and mite antigen specific IgE were found in all cases.Paraesthesia in limb extremities and positive Lhermitte sign were the main clinical features,while no optic,motor,urinary and defecation disturbance were found.Oligoclonal banding of cerebrospinal fluid and serum aquaporin 4 (AQP4) antibody were both negative in all cases.Spinal MRI showed lesions were hypointense on T1 and hyperintense on T2 at the posterior column of T2-3 segment with abnormal enhancement in case 1,hypointense on T1 and hyperintense on T2 at C2/3 segment with mild swelling in case 2 and hypointense on T1 and hyperintense on T2 at C3-5 segments with swelling and abnormal enhancement in case 3.Vitamin B were used in one patient,while the other two patients improved after the treatment with high-dose corticosteroids.Conclusions Subacute myelitis predominantly presents as paraesthesia in limb extremities with elevated serum total IgE and mite antigen specific IgE,while severe motor disorders are rare.Swelling and abnormal enhancement lesions at the posterior column of cervical cord are the common imaging features.Treatment with corticosteroids is recommended to be sustained for 3-6 months.

Objective To establish an HPLC method for the simultaneous determination of quercetin and kaempferol inKaempferia galanga L..Methods ODS2 C18 (5μm, 4.6 mm×150 mm) was used as chromatographic column; methanol-0.4% phosphate (47:53) was the mobile phase; the flow rate was 1 mL/min; column temperature was 30℃; the detection wavelength was 367 nm; the injection volume was 10μL.Results Quercetin showed good linear relationship in the range of 0.016 5–1.65μg (r=0.999 7). The average recovery rate was 96.8%, RSD=2.02%; kaempferol showed good linear relationship in the range of 0.014 6–1.46μg (r=0.999 5). The average recovery rate was 97.3%, RSD=1.77%.Conclusion The method is simple, accurate, and with good reproducibility, which can be used for content determination of quercetin and kaempferol inKaempferia galanga L..

OBJECTIVE:To establish a method for the content determination of kaempferol in Kaempferia galanga. METH-ODS:HPLC was performed on the column of Diamonsil ODS2 C18 with mobile phase of methanol-0.4% Phosphoric acid solution at a flow rate of 1 ml/min,detection wavelength was 367 nm,column temperature was 30℃,and injection volume was 10 μl. RE-SULTS:The linear range of kaempferol was 0.001 58-0.158 mg/ml;RSDs of precision,stability and reproducibility tests were low-er than 3%;recovery was 95.52%-99.32%(RSD=1.47%,n=6). CONCLUSIONS:The method is simple,accurate and reproduc-ible,and can be used for the content determination of kaempferol in K. galanga.

Objective:To establish a quality control standard for psoraleae fructus tinctures. Methods:The identification was de-tected by TLC on silica gel G plates with hexane-ethyl acetate (8∶2) as the developing solvent. An HPLC method was applied in the quantitative determination of psoralen and isopsoralen as the effective components. The analytical column was Intersil ODS-3 (250 mm × 4. 6 mm,5 μm) with methanol-water(45∶55) as the mobile phase;the flow rate was 1 ml·min-1;the detection wavelength was at 246 nm and the column temperature was set at 35℃. Results:The characteristic spots for psoralen and isopsoralen were identified by TLC. The concentration of psoralen showed good linearity within the range of 4. 88-187. 50μg·ml-1(r=0. 999 9)and the average re-covery was 102. 63% and RSD was 0. 43%. The concentration of isopsoralen showed good linearity within the range of 4. 25-163. 20μg ·ml-1(r=0. 999 9), and the average recovery was 102. 37% and RSD was 1. 13%. Conclusion: The qualitative and quantitative methods are simple, accurate, feasible and repeatable, which can be used in the quality control of psoraleae fructus tinctures.

Objective: To establish an HPLC method for the determination of coenzyme A in coenzyme complex for injection. Methods:The content determination was performed on an Intersil ODS-3 column with methanol-pH 6. 5 phosphate buffer solution (10∶90) as the mobile phase. The detection wavelength was 259 nm and the flow rate was 1. 0 ml·min-1 . The column temperature was 30℃ and the injection volume was 20 μl. Results:The linear range of coenzyme A was 1.624-32.482 u·ml-1(r=0.999 9). The average recovery was 102. 36% and RSD was 1. 14%(n=6). Conclusion: The method is simple, accurate and reproducible, and it can be used for the quality control of coenzyme complex for injection.

Diagnostics probation teaching has important value in the medical teaching for foreign students. The practice experiences of diagnostics probation teaching for foreign students in the last 3 years are reviewed from the management of teaching,practice of teaching,supervision of teaching and examine of teaching in order to improve the effect of diagnostics probation teaching for foreign students.

Objective: To investigate the innate immune response of influenza virus-infected glial cells,the transcription levels in chemokines in mouse microglia and astrocytes were detected which pre-infected by human H1N1 or avian H5N1 influenza viruses.Methods: The glial cells isolated from neonatal mice cerebral cortex were cultured and further microglia and astrocytes were purified.The primary mouse microglia and astrocytes were infected in vitro by H1N1 or H5N1 influenza viruses in a multiplicity of infection (MOI) 2.Eight hours post infection,the influenza virus nucleoprotein (NP) was detected by immunofluorescence to identify the proportion of infected cells.The cellular RNA were extracted at 6 h and 24 h to detect the transcriptional level of chemokines by semi-quantitative RT-PCR.Results: More than 95% of the microgha and astrocytes which isolated from mice were infected.The transcription levels of CCL-3,CCL-5,CXCL-2,CXCL-9 and CXCL-10 from infected microglia and astrocytes were upregulated.Futhermore,the mRNA level of CXCL-10 increased much more.In addition,avian H5N1 influenza virus could induce more stronger upregulation of those chemokines than human H1N1 did.Conclusion: The mouse microglia and astro cytes which are infected by H1N1 influenza virus or H5N1 influenza virus could induce upregulation of transcription level of chemokines.