Experimental investigation of HGF inhibiting glial scar in vitro.

To study the inhibitory effect of Hepatocyte growth factor (HGF) on the responsive hyperplasia of damaged astrocytes in vitro. We prepared damaged model of astrocytes to simulate the responsive hyperplasia of damaged astrocytes in vivo by culturing astrocytes in vitro; After the first day of Ad-HGF transfection, astrocytes were scratched, then after the first, the third, and the fifth day of scratch, we detect the expression amount of astrocytes specific glial fibrillary acidic protein (GFAP) and the ratio of S-phase cells with flow cytometry, both of which can reflect the proliferation status of damaged astrocytes; After HGF was added in scratched astrocytes, the activity of SPK and MAPK (P42/44) were detected by autoradiography and immunoblotting test; After adding different concentrations of HGF protein in astrocytes cultured in different serum concentrations and adding diverse concentrations of HGF protein, SPK and SPK inhibitor DMS in scratched astrocytes, we detect cell proliferation with 3H-TDR incorporation. The first day after Ad-HGF transfected astrocytes were scratched, the amount of GFAP secreted by astrocytes were decreased significantly (P < 0.

[Screening of the genes of hepatitis B virus e antigen interacting proteins].

Objective: To screen and clone the genes in hepatocytes which encode protein that can interact with hepatitis B e antigen(HBeAg) by yeast-two hybridization.

Methods: Recombined HBeAg bait plasmid (pGBKT7-eAg) was transformed into yeast AH l09, followed by mating with yeast Yl87 containing liver cDNA library plasmid in 2 x YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-Ade-His) which contains X-a-gal for selecting positive blue clones.

[Study on binding of HBeAg to CD81].

Aim: To investigate the interaction between HBeAg and CD81.

Methods: The CD81 gene was amplified by RT-PCR from HepG2 cells. The recombinant expression vector pGADT7-CD81 was constructed by routine molecular biological method.

[Construction and expression of DNA-binding domain plasmid with hepatitis B virus e antigen in yeast double hybrid system].

Background: Using hepatitis B virus e antigen (HBeAg) gene to construct the DNA-binding domain vector, which can express HBeAg in yeast cell, and can be used in yeast double hybrid as "bait plasmid" to look for the gene from the cDNA library, which expresses the protein that can interact with HBeAg.

Methods: PCR was performed to amplify the HBeAg gene from a sera of hepatitis B patient. The product of the amplification was inserted into T-vector and was verified by sequencing.

[The expression and activity detection of a variant N protein of SARS-CoV].

Aim: To construct an expression vector pGEX-2T/N, and to express the fusion protein consisting of N protein of SARS-CoV and GST in E.coli.

Methods: The N region gene of SARS-CoV was cloned by RT-PCR.

[The cytokine secretion of peripheral blood mononucleocytes from patients infected with HCV].

Aim: To detect the levels of cytokines secreted by PBMC from patients infected with HCV after culture for 72 hours in-vitro, so as to reflect the immune state of the HCV-infected patients.

Methods: The levels of cytokines in the culture supernatant of PBMCs were detected by ELISA.

Results: (1)Compared with the cytokine level of normal control, the levels of IFN-gamma, TNF-alpha, and IL-10 of HCV patients notably increased while IL-2, IL-4, and IL-12 were not detected in the culture supernatant of PBMCs from both normal control and HCV patients after culture for 72 hours.

[The observation of the lymphocyte phenotype change in different lymphoid tissues of mice after intranasal immunization with bivalent Shigella vaccines].

Aim: To observe the changes of lymphocyte phenotype in different lymphoid tissues of mice at various time after intranasal immunization with bivalent Shigella vaccines.

Methods: BALB/c mice were randomly divided into three groups, 30 mice per group. Mice were intranasally immunized respectively with PBS, FSM-2117 or FS-5416 four times (bacterial number was sequentially 5x10(6), 1x10(7),4x10(7)and 4x10(7)CFU/mouse) with two week intervals.

[Cloning and expressing the E2 subunit of pyruvate dehydrogenase complex].

Objectives: To construct the expression vector of the pyruvate dehydrogenase complex E2 subunit gene (PDC-E2).

Methods: The PDC-E2 gene was amplified from human lymphocytes with RT-PCR, and was cloned into pExSecI vector to induce the PDC-E2 expression. The products were identified with western blot and ELISA.