| As potent activators of T lymphocytes, SEs (staphylococcal enterotoxins) have a potential to be used in tumor therapy. Research showed that vaccination of mice with antigen followed by treatment with a combination of staphylococcal enterotoxins A and B leads to increasing in CD4 + and CD8 + T-cell populations, cytotoxic T-lymphocyte activity and interferon-γproduction, leads to significant protection against subsequent challenge with tumor cells, enhanced survival of mice. Another research shows that superantigens also have profound effects on the humoral immune responses. Injection of BSA followed by a combination of superantigens staphylococcal enterotoxin A and staphylococcal enterotoxin B enhanced the anti-BSA Ab response in mice 4-fold as compared with mice given BSA alone. But the superantigen effect was completely blocked by the CD4+ T cell inhibitory cytokine IL-10. If the tumor specific antigen is combined with SAg, more significant and specific protection against tumor should be expected.In section one, HSP65-MUC1 VNTR2 was expressed in E. coli. After identification and purification, its activity of inhibiting tumor growth was evaluated in vivo.Heat shock proteins, also called stress protein, are one kind of highly evolutionarily conserved proteins with significant physiological function. Recent reseaches showed that HSPs were closely related with tumor vaccine which could bind many intracellular peptides and induce specific anti-tumor immunity response through the receptors in APC cells. As the role of heat shock protein to cancer immunity being studied, HSP-based cancer vaccines have been one of the focuses of cancer therapy research.Muc1, as one kind of mucins, is widely expressed both by normal glandular epithelial cells and cancer cells originated from epithelial cells. MUC1 is a high molecular weight glycoprotein with a large amount of carbohydrates O-linked to the protein core. It is a transmembrane protein with a unique extracellular domain consisting mainly of variable number of tandem repeats (VNTRs) which is made up of 20 amino acids. The unique characteristics of MUC1 in many types of epithelial cancers, such as excessive expression, aberrant cellular distribution and underglycosylation-induced exposure of the normally cryptic core protein, have made MUC1 a promising candidate antigen in cancer immunotherapy. The PDTRP epitope of MUC1 core protein is promising anti-tumor molecular because it could not only be recoginized by MUC1 antibody, but also be recognized and killed by cytotoxic T lymphocytes (CTLs) without restriction of MHC.Firstly MUC1 VNTR2 were generated by gene splicing and sub-cloned to pET28a(+) to construct the recombinant expression vector HSP65-MUC1 VNTR2-pET28a(+). E. coli BL21(DE3) bearing the plasmid was induced with IPTG for protein production. Target protein was expressed stably in the soluble fraction of bacterial extract. Western blot with monoclonal antibody was positive. HSP65-MUC1 VNTR2 was purified by Q-Sepharose ion-exchange chromatography and gel filtration to above 95% purity. To investigate whether HSP65-MUC1 immunization could inhibit the growth of MUC1-expressing B16 tumor in C57BL/6 mice, we first tested the dosage response of HSP65-MUC1 in a pilot study. We found that tumor inhibition rate of immunization with 2.5μg/each could reach 72.08%, with very significant difference with PBS group. The tumor inhibition rate of 10μg/each group was 37.29%,but without significant difference with PBS group. Although both the high and the low dosage could inhibit the tumor growth, yet the tumoricidal effect of low dosage group was greater than that of the high one. The result indicated that HSP65-MUC1 could induce a potent anti-tumor CTL response, inhibit the tumor growth. It can be used in the future research as the cancer vaccine.Although SAg showed profound effect on T cell, yet the amount of tumor specific CTL was less in T cell pool activated by SAg. So if the tumor specific antigen is combined with SAg, more tumor specific CTL should be expected. In Section two, in order to evaluate if SEA/SEB can enhance the HSP65-MUC1 VNTR2 specific cellular immune response, C57BL/6 mice were devided into HSP65-MUC1 VNTR2+SEA/SEB group, HSP65-MUC1 VNTR2 group, SEA/SEB group and PBS group, respectively. After immunity, lymphocytes from murine spleen were obtained and pulsed with peptide MUC1 VNTR2. The result showed that the MUC1-specific CTLs could be efficiently induced by HSP65-MUC1 VNTR2+SE group and HSP65-MUC1 VNTR2 group and the specific lysis rate was about 36%,30%respectively. The ELISPOT assay showed that the number of T cell that secreting IFN-γof HSP65-MUC1 VNTR2 +SEA/SEB group was the highest, about 81.22, two times more than HSP65-MUC1 VNTR2 group, with the significant difference with PBS group.The results in vivo also showed that SEA/SEB could enhance the tumoricidal efficacy of HSP65-MUC1 VNTR2. Prophylactic immunization with HSP65-MUC1 VNTR2 induces growth inhibition of subsequently implanted tumors in HSP65-MUC1 VNTR2+SEA/SEB group. The tumor inhibition rate is about 62%, with significant difference with PBS group. The other three groups, HSP65-MUC1 VNTR2, SEA/SEB and PBS group, didn't efficiently inhibit tumor growth. In the study of the prophylactic effect of HSP65-MUC1 to MUC1 negative B16 tumor, CTL from HSP65-MUC1-immunized mice did not inhibit B16 tumor growth effectively. In the study of the therapeutic effect of HSP65-MUC1, only the tumor inhibition rate of HSP65-MUC1 VNTR2+SE group has the significance with PBS group. On day 58, the mice of HSP65-MUC1 VNTR2+SE group were still alive. The result in vitro and in vivo showed that SEA/SEB could enhance the HSP65-MUC1 VNTR2 specific cellular immune response.
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