AIM: To investigate the immune mechanisms for Periplocin from Cortex Periplocae (CPP) in tumor-bearing mice. METHODS: H_(22) tumor-bearing model BALB/c mice were applied to evaluated in vivo immunoregulatory effect of CPP. The influence of different dose CPP (0.25, 0.50 and 1.00 mg/kg) on immune organs in tumorbearing mice were observed. T cell subsets of mice spleen were detected by flow cytometry. MTT assay was used to determine the influence of CPP on lymphocyte proliferation of mice spleen stimulated by ConA. The levels of TNF-α, IL-2 and IL-12 in serum from mice were detected by means of ELISA. RESULTS: Thymus index and spleen index of H_(22) tumor-bearing model control mice became less than that of normal mice (P < 0.05). Compared to both model and normal control groups, thymus index and spleen index of H_(22) tumor-bearing mice treated with CPP increased obviously (P < 0.05). CPP had no influence on the number of CD8~+ T cells, but up-regulated markedly the number of CD3~+, CD4~+ T cells and the ratio of CD4~+/CD8~+ in tumorbearing mice. In CPP-treated mice, the percentage of CD3~+, CD4~+ T cells were not different from normal mice (P<0.05), the ratio of CD4~+/CD8~+ was higher than that of normal mice (P < 0.05). CPP enhanced obviously lymphocyte proliferation of mice spleen induced by ConA, the SI scores were even higher than that of nornal mice. The levels of TNF-α, IL-2 and IL-12 in serum from CPP-treated mice, increased significantly compared to model control group (P<0.05) in a dose-dependent manner, were similar to or higher than that of normal mice. CONCLUSION: CPP protected immune organs of tumor-bearing mice, increased obviously the percentage of CD4~+ and CD4~+/ CD8~+ among the T cell line, and enhanced lymphocyte proliferation of mice spleen significantly, stimulated the production of TNF-α, IL-2 and IL-12. The results suggested that CPP possessed potent immunoregulatory effect.

AIM: To construct human Egr-1 promoter luciferase reporter system and study its activity induced by i-onizing radiation. METHODS: Egr-1 promoter was obtained by human genomic PCR and cloned into pGL3-basic vector. After transfection of recombinant plasmid into human tumor cells, the Egr-1 promoter activity induced by ionizing radiation was detected by luciferase reporter assay. RESULTS: The luciferasy reporter system of Egr-1 promoter was successfully constructed. The activity of Egr-1 promoter was substantially increased after different doses of IR and reached to the peak at the time point of 48h after IR. CONCLUSION: The Egr-1 promoter was constructed in this study showed IR inducible activity in tumor cells, laying foundation for the research of radiation, mediated gene therapy.

The human immunodeficiency virus type 1 (HIV - 1 ) envelope glycoprotein gp41 plays a critical role in the fusion of viral and target cell membranes. The gp41 extracellular domain,which contains fusion peptide (FP), N - and C - terminal hydrophobic heptad repeats (NHR and CHR, respectively).Peptides derived from NHR and CHR regions,designated N- and C- peptides, respectively, can interact with each other to form a six - stranded coiled - coil domain, representing the fusion-active gp41 core. Our previous studies demonstrated that the C- peptides have potent inhibitory activity against HIV- 1 infection.These peptides inhibit HIV- 1 -mediated membrane fusion by binding to NHR regions for preventing the formation of fusion- active gp41 core.One of the C - peptides, T - 20, which is in the phase Ⅲ clinical trails, is expected to become the first peptide HIV fusion inhibitory drug in the near future.However, this peptide HIV fusion inhibitor lacks oral availability and is sensitive to the proteolytic digestion.Therefore, it is essential to develop small molecular non -peptide HIV fusion inhibitors having similar mechanism of action as the C- peptides.We have established an ELISA- based screening assay using a unique monoclonal antibody, NC- 1, which can specifically bind to a conformational epitope on the gp41 core domain.Using this screening assay, we have identified a small molecular anti- HIV- 1 compound,named ADS-Jl, which inhibits HIV- 1- mediated membrane fusion by blocking the interaction between the NHR and CHR regions to form the fusion - active gp41 core.This compound will be used as a lead to design and develop novel HIV fusion inhibitors as new drugs for the treatment of HIV infection and/or AIDS.

AIM: To detect the expression of the Calreticulin and HPV E2 Fusion Protein in B16, and study the effects on proliferation and apoptosis of B16 cell lines in vivo. METHODS: To construct eukaryotic fluoresce expression vector pEGFP-CRT-E2, pEGFP-CRT and pEGFP-E2. Then the recombinant plasmids were transfected into B16 cells by Lipofectamine 2000. The expression of proteins was detected by Western blot. The location of different GFP fusion proteins in B16 was tested by inverted fluoresce microscope. Flow cytometry was applied to detect the effects of fusion proteins on the growing of B16 and then the apoptosis effects of B16 induced by different proteins were observed. RESULTS: The correctly constructed recombinant plasmid pEGFP-CRT-E2 and the expression of CRT-E2 gene could be detected in B16 cells. Apoptosis of B16 cells could be detected after the transient transfection. Meanwhile, the apoptosis rate of B16 cells transfected by pEGFP-CRT-E2 was much higher than that of control cells. And cell cycle G1/G0 arrest was also found (P < 0.01). CONCLUSION: The eukaryotic expression plasmid pEGFP-CRT-E2 is successfully constructed and it could correctly express the fusion protein in B16 cells. And the B16 cells transfected by plasmid pEGFP-CRT-E2 could induce apoptosis.

AIM: To explore method of stimulating murine bone marrow-derived dendritic cells by lipopolysaccharide(LPS), transforming growth factor-β1 (TGF-β1)and to study their biological character. METHODS: Murine bone marrow-derived dendritic cells were cultivated with cytokine GM-CSF and IL-4 for 6 days, BMDC was stimulated by control, LPS, TGF-β1, LPS +TGF-β1 for 48 hours respectively. Morphological characters of BMDC were observed by a inversed microscope, surface molecules such as CD_(11C), CD_(80), CD_(86)and MHC Ⅱ were detected by flowcytometry, Interleukin-6 and interleukin-12 p70 in co-culture medium was quantified by ELISA. RESULTS: In LPS group it presented the most typical DC morphology with the highest expression of CD_(80), CD_(86) and MHC Ⅱ, the strongest ability in mixed lymphocyte reaction, higher level of IL-6 and IL-12 p70 compared with control, TGF-β1, LPS + TGF-β1 ( P < 0. 05). While in TGF-β1 group it presented the less typical DC morphology with the lower expression of CD_(80), CD_(86), MHC Ⅱ, weaker ability in mixed lymphocyte reaction, and lower levels of IL-6 and IL-12 p70 compared with control and LPS (P < 0.05). CONCLUSION: LPS can stimulate maturation of BMDC in its late differentiation which makes it presents a more significant biological characteristics. TGF-β1 can inhibit maturation but not differentiation of BMDC thereby can prevent its biological characteristic presentation.

Aim To investigate the signaling basis of the biological effects of IL-6 in U937 cells. Methods We determined the effect of IL-6 on the growth and differentiation of U937 cells and the effect of IL-6 on the activation of nuclear factors in U937 cells. Results IL-6 induced macrophage differentiation of U937 cells. Differentiated cells had strengthened ANAE (acid naphthyl acetate esterase)and NBT(nitroblue tetrazolium)-reducing activities and CD54 expression was upregulated. STAT3 could be remarkably activated by IL-6 in U937 cells and STAT3 activity was dependent on dose and time course of IL-6 treatment. However, NF-IL6,NF-κ B, AP-1 and other STAT members were not activated by IL-6. Conclusion IL-6 might induce terminal differentiation of U937 cells via JAK-STAT3 pathway.

To develop simple, rapid, and efficacious diagnostic methods for malaria is one of the remaining key tasks for malaria control. Previously, we have created a phage-displayed antibody library against Plasmodium falciparum. Six clones of antibody with good reactivity to HRP-II in ELISA were isolated from the library after 3 rounds of enrichment. Soluble ScFvs were produced and the characteristics were determined. The results of Western blot showed that they could bind to HRP-II specifically and had a relative molecular mass(Mr) about 31 000. The work provided a solid fund for diagnostic kit development for malaria.

AIM: To investigate the significance and function of IFN-γ on the changes of peripheral blood platelet count during tumor-rejection induced by a low dose of melphalan in C57BL/6 mice. METHODS: Mouse tumor rejection model induced by a single dose of melphalan was used in this experiment. Different gene-type tumor-bearing mice (IFN-γ~(+/-) and IFN-γ~(-/-)), which had the same genetic background of C57BL/6, were treated intraperitoneally with melphalan (7.5 mg/kg). Tumor size was observed and recorded every one to three days in these different gene-type mice subsequently. Blood samples were obtained from orbital venous sinus on different days before and after melphalan treatment, and then complete blood counts were performed. The function of IFN-γ on the efficacy of chemotherapy and the changes of blood platelet count in IFN-γ~(+/-) and IFN-γ~(-/-) mice after melphalan treatment was analyzed. RESULTS: There was no significant difference in tumor sizes and blood platelet count between IFN-γ~(-/-) and IFN-γ~(+/-) mice (P>0.05). On the first day after melphalan (7.5 mg/kg) treatment, there were no significant changes in tumor sizes between mice in these two groups (P>0.05). Tumors shrank a little in IFN-γ~(-/-) mice and then grew gradually. Tumors relapsed in 2 w after melphalan injection in all IFN-γ~(-/-) mice, while tumor volumes decreased progressively and tumor cured at last in IFN-γ~(+/-) mice. The number of blood PLT in IFN-γ~(+/-) mice increased to (1935±378)×10~9/L 6 h after melphalan treatment, significantly higher than before (P<0.01); While in IFN-γ~(-/-) mice it was (1183±186)×10~9/L 6 h after melphalan treatment, no obvious increase than before. There was significant difference in blood PLT 6 h after melphalan treatment between IFN-γ~(+/-) and IFN-γ~(-/-) mice (P<0.01). Later, the numbers of blood PLT in IFN-γ~(+/-) mice decreased gradually and it dropped to normal (1158±270)×10~9/L on 11th day after melphalan treatment (P>0.05); While it sustained in normal range in IFN-γ~(-/-) mice. There was no significant difference in blood platelet count between IFN-γ~(-/-) and IFN-γ~(+/-) mice. CONCLUSION: Peripheral blood platelet count increased on the first day after melphalan treatment and tumors cured in IFN-γ~(+/-) mice; While tumors relapsed and there is no increase in blood platelet count on the first day after melphalan treatment in IFN-γ~(-/-)mice. These data indicated that the increase of blood PLT count was related to the function of IFN-γ in tumor-bearing mice in vivo during tumor rejection induced by a low dose of melphalan.

AIM:To construct eukaryotic expression vector of Sirt2 and detect its expression in HEK293 cells. METHODS: Total RNA was isolated from brain tissue of a-dult SD rat. A 1 130 bp fragment containing the coding region of Sirt2 was amplified by RT-PCR and the resulting PCR product was subcloned into PMD20-T vector and se-quenced. Coding region of Sirt2 was generated with PCR by using the PMD20-T-Sirt2 as template, the amplified PCR fragment was inserted into the EcoR I and Hind Ⅲ sites of the pcDNA3. 1myc-his(-)A expression vector, and the sequence was confirmed by DNA sequencing. The expression of new construct pcDNA3.1 myc-his(-) A-Sirt2 in HEK293 cells was detected by immunofluorescence. RESULTS: The full length coding region of Sirt2 was obtained and confirmed by sequencing, the expression of Sirt2 was detected successfully in HEK293 cells. CONCLUSION: The eukaryotic expression vector of Sirt2 has been successfully constructed, which will provide a useful tool for designing an in-depth investigation of the role of Sirt2.

Aim To express hepatitis C virus glycoprotein E (gE) deleting carboxy-terminal 31 amino acids, and detect anti-E antibody in HCV patients using expressed gE. Methods E gene derived from HCV H strain was inserted into baculovirus transfer vector containing a polyhedrin promotor. The recombinant plasmid was cotransfected into insect cell sf9 with a viral expression vector. The expression of gE was analyzed with Western blot, and the cells were used for dectecting antibodies against E1 and E2 in 35 hepatitis C patients by indirect immunofluorescence. Results A series of proteins with different relative molecular masses(Mr) could be detected by Western blot. Results from indirect immunofluorescence staining showed and only 4 patients were anti-E antibody positive gE was located in cytoplasm. Conclusion HCV gE is expressed successfully in insect cells, the study lay the foundation for further developing HCV vaccine.