ABSTRACT
@#Objective: To prepare the fusion protein mGM-CSF-GnRH3 (mGGn) of mouse granulocyte-macrophage colony stimulating factor (mGM-CSF) combining with gonadotropin releasing hormone (GnRH) and the fusion protein mGM-CSF-GRP6 (mG6) of mGM-CSF combining with gastrin-releasing peptide (GRP), and to investigate the inhibitory effect of the above two fusion proteins on B16F10 melanoma in vitro as well as to preliminarily predict their isoelectric point, relative molecular weight, hydrophobicity, stability, subcellular localization, signal peptide, spatial structure and potential epitopes. Methods:After the successful preparation of mGGn and mG6, the effects of different concentrations of fusion proteins on tumor cell morphology, migration, proliferation and cell cycle were detected by microscopic observation, scratch test, CCK-8 method and flow cytometry, respectively. The protein online analysis systems EXPASY, GOR4, SWISS MODEL were used to predict the basic properties and secondary/tertiary structure of recombinant fusion proteins. The B cell epitopes were predicted by IEDB and ABCpred software, the CTL epitopes were comprehensively predicted by SYFPEITHI, BlMAS and NetCTL software, and the Th epitopes were predicted by NetMHCIIpan 3.1 Server and IEDB software. Results:Both mGGn and mG6 inhibited the migration and proliferation of tumor cells. mGGn could block B16F10 cell cycle at G1 phase while mG6 could block B16F10 cell cycle at S phase, all of which prevented cells entering into G2 phase to inhibit tumor cell growth. The mGGn and mG6 fusion proteins got diverse structures and had multiple potential B epitopes, CTL epitopes and Th epitopes. Conclusion: mGGn and mG6 have inhibitory effect on B16F10 melanoma in vitro, and bioinformatics predictions have laid a foundation for further study of the biological functions and immunological activities of these fusion proteins.
ABSTRACT
This study aimed at investigating the inhibitory effects and the anti-tumor mechanisms of co-adminis-tration of fusion proteins mGM-CSF/βhCG ( GC ) and hVEGF121/βhCG ( VC ) on RM-1 prostatic cancer and B16 F10 melanoma in the C57 BL/6 J mouse model. Two recombinant stains containing pET-28 a-mGM-CSF-X10-βhCGCTP37 and pET-28 a-VEGF-M2-X10-βhCG-CTP37 were induced by lactose to express fusion proteins. The fusion proteins were separated and purified to prepare the anti-tumor protein vaccines ( VC protein vaccine and GC protein vaccine) , which were then mixed to prepare a combined protein vaccine named VGC protein vac-cine. The prostatic cancer and melanoma tumor-bearing mice C57 BL/6 J were immunized with described vac-cines, then the growth of each tumor was measured;splenocyte proliferation of immunized mice was detected;and the cytotoxic effects of the vaccine on tumor cells were tested. After that, the in vivo concentrations of IFN-γ and anti-hVEGF antibodies were investigated by ELISA. The difference between each experimental group and normal saline group ( NS) was statistically significant in both tumor-bearing mouse models ( P <0. 05) respectively. Besides, VGC group exhibited significantly better anti-tumor effect compared with the GC and VC groups with the anti-tumor rate ( 41. 7 ± 0. 83)% and ( 46. 4 ± 1. 27)% for prostatic cancer and melanoma tumor, respectively. The co-administration of the two proteins, VC and GC, could inhibit the growth of RM-1 prostatic tumor and B16F10 melanoma effectively via anti-tumor immunity and anti-tumor angiogenesis.
ABSTRACT
@#An expression vector pET-28a-mGM-CSF-X10-βhCGCTP37 plasmid containing the βhCG and mGM-CSF gene was designed and constructed. The fusion protein was induced by lactose and purified by ammonium sulfate precipitation and DEAE-cellulose anion exchange column. Then dendritic cells(DC)in C57BL/6J mice were extracted and sensitized by the fusion protein to obtain DC vaccine. The DC vaccine was inoculated to C57BL of / 6J mice with prostate cancer RM-1. The results indicated that the anti-tumor effects of DC group and DC combined with paclitaxel(DP)group were superior to that of paclitaxel(Pac)group(P< 0. 01), and the anti-tumor effect of DP group was better than that of DC group. Thus, the constructed DC vaccine can inhibit the growth of prostate cancer, and have synergistic anti-tumor when used with paclitaxel.
ABSTRACT
BACKGROUND: There are many growth media for cultivation of human melanocytes (MGM), depending on the supplements added, and the growth of cells is closely related to these components. To understand melanocytes in vivo, it is necessary to find out the biological or biochemical characteristics of melanocytes grown in physiologic growth medium (P-MGM) and phorbol-12-myristate 13-acetate (PMA)-containing medium (C-MGM). OBJECTIVE: To investigate the expression of different biochemical markers of melanocytes grown in C-MGM and in P-MGM. METHODS: C-MGM is basically composed of PMA (10 ng/ml), and bFGF (3 ng/ml), and is now commercially available for melanocyte culture. P-MGM is a physiologic growth medium containing physiologic mitogens such as bFGF (10 ng/ml), ET-1 (10 nM), and alpha-MSH (12 nM). The cell proliferation and the expression of biochemical markers were measured in cultured human melanocytes which were grown in either C-MGM or P-MGM. RESULTS: In this study, there was significant difference in cell proliferation between cells grown in C-MGM and P-MGM (p<0.01). The tyrosinase activity and melanin contents were significantly increased in C-MGM. The expression of TRP1, MART-1 and p53 in mRNA level was higher in C-MGM than in P-MGM. The up-regulation of p53 protein expression was also observed in C-MGM. CONCLUSION: The proliferation and expression of p53, at both transcriptional and translational levels were increased when melanocytes were grown in C-MGM, compared to P-MGM. This data suggests that p53-mediated melanization is to some degree related with phorbol ester, and should further be elucidated.
Subject(s)
Humans , alpha-MSH , Biomarkers , Cell Proliferation , Melanins , Melanocytes , Mitogens , Monophenol Monooxygenase , RNA, Messenger , Up-RegulationABSTRACT
Objective:hIL 2/mGM CSF fusion gene was constructed and expressed in E.coli.recombinant hIL 2/mGM CSF fusion protein had both biological activity of hIL 2 and mGM CSF.Methods:The hIL 2 and mGM CSF genes were amplified by PCR (splicing by overlap extension,SOE) and ligated with two prolines linkers.Then the fusion gene was cloned into vector pLY4 and PBV220.The recombinant plasmid were transfected into E.coli and expressed.Results:The sequence of hIL 2/mGM CSF fusion gene was correct the hIL 2/mGM CSF fusion protein was highly expressed in E.coli and that comprises 20% of total bacterial protein.The results of biological activity assay showed that the expressed product possessed both of the activities of hIL 2(4.5?10 5 U/mg) and mGM CSF(3.85?10 6 U/mg).Conclusion:Have successful constructed the recombinant hIL 2/mGM CSF fusion protein which possessed bioactivities similar to that of the natural hIL 2 and mGM CSF.
ABSTRACT
Objective: To investigate the vaccine potency of GM-CSF anchored B16 tumor cells. Methods: In this study, mGM-CSF was expressed on surface of B16 mice melanoma cells by GPI-modifying. C57BL/6 mice were inoculated with GM-CSF anchored cells and wide-type B16 cells to evaluate whether the GM-CSF anchored cells could elicit a protective and systemtic antitumor response. Results: GM-CSF anchored cells resulted in remarkable loss of tumorgenicity in syngenetic mice. The tumor occurrence rate of GM-CSF anchored B16 cells was 58. 8% on C57BL/6 mice with 1 ? 106 B16 cells/mice inoculated ( n = 12) and that of wide-type B16 cells was 100% , The C57BL/6 mice receiving inoculation with 5 ? 105GM-CSF anchored cells/mice never grew tumor. These mice were challenged with wide-type B16 cells, and only a minority of mice grew tumor after wide-type B16 cells inoculation. Conclusion:GM-CSF protein anchored cells could elicit a protective and systemtic antitumor responses.