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Objective:To investigate the MUC1 specific immune response enhanced by thmosinα1(Tα1)using MUC1-MBP as the specific antigen,and to discuss the feasibility of MUC1-MBP as an adjuvant.Methods:The C57BL/6 mice were randomly divided into normal saline group,MUC1-MBP+BCG group and MUC1-MBP+Tα1 group.The T cellular immune activities,MUC1 special antibody and subclass,anti-tumor effect of Tα1 combined with MUC1-MBP were detected.4-7 d after the 3rd immunization,the spleen indexes of the mice in various groups were measured;the lymphocyte proliferation response was used to detect the stimulate index(SI)of the mice in various groups;the levels of specific cytokines IFN-γ,IL-2,IL-4 and IL-10 in the supernatant of spleen cells of the mice were detected by ELISA;the level of serum MUC1-specific antibody was detected by ELISA.The C57BL/6 mice were inoculated with B16-MUC1 7 d after the last immunization and the survival of the mice was observed.Results:Compared with normal saline group,the spleen index and SI of the mice in MUC1-MBP+Tα1 and MUC1-MBP+BCG groups were significantly increased(P< 0.05 or P<0.01);the specific SI of MUC1 were significantly increased(P< 0.05).Compared with normal saline group,the levels of IFN-γ and IL-2 in the supernatant of spleen cells of the mice in MUC1-MBP+BCG group were obviously increased(P<0.05);the levels of IL-4 and IL-10 were slightly increased,but there were no significant differences(P>0.05);compared with normal saline group,the level of IL-4 in MUC1-MBP+Tα1 group was obviously increased(P< 0.01);the levels of IFN-γ,IL-2 and IL-10 were slightly increased,but there were no significant differences(P>0.05).The titer of MUC1 specific antibody was increased with the increase of concentration of Tα1.The antibody subtype detection results showed that compared with normal saline group,the level of IgG1 in MUC1-MBP+BCG group was significantly increased(P< 0.05 or P< 0.01),and the level of IgG2a had no obvious change.The tumor prevention experiment results showed that compared with normal saline group,the survival rates of the mice in MUC1-MBP+BCG group and MUC1-MBP+Tα1 group had no significant differences.Conclusion:MUC1-MBP combined with Tα1 prefers to Th2 immune responses in the mice.It indicates that Tα1 can be used as an adjuvanted preventive vaccines,but not suitable for therapeutic vaccines.
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Objective:To study the inductive effect of recombinant MUC1-MBP fusion protein combined with R848 on the immune activity of T cells in the mice,and to provide experimental basis for searching the suitable adjuvants of recombinant MUC1-MBP fusion protein.Methods:A total of 21 C57BL/6 mice were randomly divided into control group(treat with normal saline),MUC1-MBP+R848 group (treated with MUC1-MBP+R848),and MUC1-MBP+baillus calmette guerin(BCG) group(treated with MUC1-MBP+BCG)(n=7).After immunized for 4-7 d,the spleen tissue was taken and the spleen indexes of the mice in various groups were measured;the stimmulus index(SI) of the mice was detected by lymphocyte proliferation response;the levels of tumor necrosis factor-γ(TNF-γ) and interleukin-4(IL-4) were detected by ELISA method;the proprotions of T lymphocyte subsets in spleen cells were analyzed by flow cytometry.Results:Compared with control group,the spleen indexes,SI,and TNF-γ levels of the mice in MUC1-MBP+ R848 and MUC1-MBP+BCG groups were significantly increased (P0.05).Compared with control group,the proprotions of CD3+T,CD4+T,and CD8+ T cells of the mice in MUC1-MBP+ R848 group and MUC1-MBP+BCG group were significantly increased(P<0.05 or P<0.01).Conclusion:Both MUC1-MBP fusion protein combined with R848 and BCG can induce the Th1 type of immune activity in the mice,and R848 is the potential candidate adjuvant for MUC1-MBP.
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Objective To evaluate the effectiveness of ultrafiltration technology in endotoxin removal from purified recombinant MUC1-MBP fusion protein (MUC1-MBP)and to demonstrate the effect of ultrafiltration on endotoxin removal.Methods CM Sepharose FF weak cation exchange (CM)(CM group), CM combined with Phenyl Sepharose 6 FF exchange (C6)(CM+C6 group),CM combined with ultrafiltration (CM+ultrafiltration group), and CM combined with C6 and ultrafiltration (CM+C6+ultrafiltration group)were used to purify the MUC1-MBP from E.coli. and remove endotoxin;the expression level of endotoxin was detected by Chromogenic End-point Tachypleus Amebocyte Lysate.Results There was a single band at the expected molecular weight of 62 000 by SDS-PAGE analysis.and the purity>96% by Quantity One analysis.The endotoxin levels in CM group and CM +C6 group were quite high and there was no significant difference between two groups (P>0.05 );the endotoxin level in CM+ultrafiltration group was significantly lower than that in CM group, and there was significant difference (P0.05 ). Conclusion The effects of CM or CM combined with C6 on endotoxin removal are quite poor, especially C6;CM combined with ultrafiltration are quite effective on endotoxin removal,and ultrafiltration plays an important role in endotoxin removal.
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New tumor antigens are continuously being discovered due to the improvement of immunologic techniques.Whether dendritic cells induce immune activation or immune inhibition after they capture tumor antigens depends on the danger signals(GM-CSF,MCP1,and HSP) or the inhibitory signals(TGF-?,IDO,and iNOS) released by tumor cells.Under the regulation of danger signals,dendritic cells activate Th1 immune response and eliminate tumors;under the regulation of inhibitory signals,they activate Th2 immune response and can not effectively eliminate tumors.Progress in tumor immunotherapy mainly is manifested by antibody-based therapy,T cell-based therapy,and tumor vaccine-based therapy.To day at least 7 antibodies have been confirmed effective when combined with chemotherapeutic agents in treatment of tumors.Despite of the progress made in antibody therapy,discovery of new targets,development of new antibodies,and expanding of the application scope to more tumors still need intensive research efforts.The clinical effects of T cell-based therapy have not been satisfactory;most tumor vaccine-based therapies are in phase Ⅰ and Ⅱ clinical trial,and the outcomes of few phase Ⅲ clinical trials are not satisfactory,leaving more work to be done for improvement.As we understand more about the roles of antibody in immune surveillance,it will help to make immunotherapy of tumors a promising strategy.
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Objective:To study MUC1 based cancer vaccine.Methods:MUC1 gene was inserted into pMAL-p2 vector and constructed recombinant pMAL-MUC1. MUC1-MBP fusion protein expression was induced by IPTG in E coli DH5? transformed by the recombinant pMAL-MUC1 .The fusion protein was analyzed by Western blot and purified by amylose affinity chromatography. The antiserum,T cell proliferation and CTL activity of spleen from C57 mice immunized by MUC1-MBP were determined respectively by ELISA,adding 3H-TdR and MTT.Results:Had successfully constructed pMAL-MUC1 expression vector,and purified MUC1-MBP and MBP. C57 mice immunized by MUC-MBP generated MUC1 specific antibody and CTL.The titer of polyclonal antibody to MUC1 was about 1∶5 760?3 221. CTL cytotoxicity to the MCF7 and lewis lung cancer cells respectively were at 47.7%?4.3% and 67.5%?6.5%.Conclusion:Human recombinant MUC1-MBP fusion protein activated T and B cell response in mice.The results suggested that the recombinant Muc1 may be used to develop protein vaccine against carcinoma.
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Objective: To study the anti-tumor effect of recombinant human MUC1-MBP. Methods: The C57BL/6 mice were in oculated with MUC1-MBP by subcutaneous. MUC1 specific CTL activity of spleen were determined by MTT; The effects on prevention and treatment of tumor were observed by establishing lewis lung cancer-carrying mice. Results: The cytotoxicity of CTL from immunized mice to the MCF7 and Lewis lung cancer cells respectively was (47.7?4.3) % and (67.5 ?6.5) %; 5?10 5 lewis lung cancer cells following immunization were injected iv into C57BL/6 mice, after three weeks, the number of lung and tail tumor colonies was 51 and 5 for PBS and MUC1-MBP groups respectively and the suvival time was significantly delayed in immunized mice. The average volume of tumors in mice with MUC1-MBP was 386 mm 3 wherea control group was 4 000 mm 3 at tumor treating experiment. Conclusions: Recombinant human MUC1-MBP have significantly effects on prevention, treatment and inhibiting metastases of tumor. Our results suggested that the recombinant MUC1-MBP might be used to develop protein vaccine against human carcinoma.