Vaccination With Recombinant Attenuated Salmonella Carrying 4-1BB Ligand Gene Affect The Growth Of Experimental Colorectal Cancer

Part one:The construction and functional identification of the recombinant eukaryotic expression vector carrying rat 4-1BBL geneObjective To investigate the effect of rat 4-1BBL upon immunoactivity and surface marker of dendritic cells(DCs) derived from bone marrow.Methods Plasmid pIRES2-EGFP-4-1BBL was constructed and tansfected into HepG2 cells by lipofectin-mediated method,and positive cells were screened by G418.The expression of 4-1BBL in cells were detected by RT-PCR,and the expression of GFP was detected by fluorescence microscopy.HepG2 cells were co-cultured with DCs for 2 days,and then the expression of MHC-Ⅱand CD80 on DCs were detected by FCM,and IL-6 and IL-12 concentrations in supernatants secreted by DCs were tested by ELISA.Results The recombinant pIRES2-EGFP-4-1BBL vector was successfully constructed and HepG2 cells transfected with pIRES2-EGFP-4-1BBL could steadily express 4-1BBL.Compared with HepG2 cells transfected by pIRES2-EGFP plasmid,recombinant HepG2 cells could markedly simulate the expressions of MHC-Ⅱand CD80 on DCs and the secretion of more IL-6 and IL-12(P＜0.05).Conclusions The recombinant rat 4-1BBL could promote the maturity of DCs derived from bone marrow.Part two:Construction and indentification of recombination Attenuation Salmonella typhimurium vaccine containing pIRES2-EGFP-4-1BBL vectorObjective:To develop strain SL3261(Attenuation Salmonella typhimurium) vaccine containing rat 4-1BBL gene eukaryotic expression vector.Methods pIRES2-EGFP-4 -1BBL vector was converted to LB5000(Salmonella typhimurium) for methylation then modified vector was electrotransfered to final host SL3261,screened single colony by antibiotics was tested by plasmid extract and PCR.Meanwhile reconstructed SL3261 and its predecessor were co-cultured with HepG2 cells to detect their invasion.Fluorescence microscope observation and RT-PCR were exploited to detect the expression of GFP and transcription of 4-1BBL.Results Similar plasmid to origin was extracted from Strain SL3261 imported with the pIRES2-EGFP-4-1BBL plasmid,which containing the interesting gene sequence about 930 bp identified by PCR.The potential of SL3261 to invade HepG2 cells was 201±46 CFU/200 HepG2 cells,and that of SL3261-pIRES2-EGFP was 163±37 CFU/200 HepG2 cells and that of SL3261-pIRES2-EGFP-4-1BBL was 158±32 CFU/200 HepG2 cells.Expression of GFP in HepG2 cells infected with SL3261 containing vector could be viewed,and the transcription of interesting gene could also be detected by RT-PCR with a 930bp strap in agarose gel.Conclusions Attenuation Salmonella typhimurium SL3261 containing recombined eukaryotic expression plasmid pIRES2-EGFP-4-1BBL was successfully constructed which can deliver recombined plasmid into HepG2 cells.Part three:4-1BB ligand Oral DNA vaccine enhancesimmunity of ratsObjective:4-1BBL gene were infected into antigen-presenting cells(APCs) in vivo by the vectors of Attenuation Salmonella to detect the effects of 4-1BBL on the immunological function of rats.Methods:Recombinant vaccine bacteria containing 4-1BBL(SL3261+ recombined plasmid) were fed on Sprague-Dawley(S-D) rat with 3 times in 2 weeks,and bacteria only containing blank plasmid(SL3261+ plasmid),blank bacteria and PBS were all kept as controls.Fourteen days after the final irrigation,rats were killed,the transcriptions of GFP in spleen cells were detected by RT-PCR,and the expressions of 4-1BBL in spleen tissues were detected by immunofluorescence staining, and the level of phenotype change in peripheral blood cells and spleen cells was determined by flow cytometry.The contents of IFN-γin supernatant secreted by spleen cells stimulated by PHA were detected by ELISA.Results:The recombinant vaccine bacteria containing 4-1BBL could successfully carry foreign gene into peripheral blood cells and spleen cells,and resulting in transcriptions and expressions in vivo.There were significantly increased expression of 4-1BBL and 4-1BB in peripheral blood cells and spleen cells(P＜0.05),up-regulation of CD3~+,CD3~+ CD8~+,and CD3~+ CD25~+ in PBC and CD3~+CD8 ~+,CD3~+CD25~+ on splenocyte were detected(P＜0.05).IFN-γreleased by PHA-stimulated splenocytes increased significantly by the vaccine as compared with controls(P＜0.05).Conclusion:these vaccines can achieve APCs 4-1BBL gene transfered in vivo,which is a promising adjuvant for the body to enhance immune function.Part four:Immune change in rats with experimental colorectal cancerObjective:to develop a model of colorectal cancer and to study the changes of immune function in this carcinogenic process.Methods:Colorectal cancer was induced consistently in experimental Sprague-Dawley(S-D) rats by the carcinogen 1, 2-dimethylhydrazine(DMH).Respectively 16 weeks,21 weeks after the tumorgenensis, the peripheral blood cells and 21 weeks after the tumorgenensis the spleen cells were all detected for the expressions of CD3,CD4,CDS,CD25,4-1BB and 4-1BBL,and the secretion of IFN-γin supernatant by spleen cells stimulated by PHA were detected by ELISA.Results:the tumorgenensis rata was 100%in all five rats.Sixteen weeks after tumorgenesis,there were down regulations of CD3~+ and CD3~+CD4~+ in peripheral blood of induced group comparing to that of control groups were,but there was no significant difference(P＞0.05);however,twenty weeks later,there were significant downregulations of CD3~+ and CD3~+CD4~+ in peripheral blood and spleen comparing to control groups (P＜0.05).IFN-γreleased by PHA-stimulated splenocytes decreased significantly by the DMH induced as compared with controls(P＜0.01).Conclusion:They were close models of human colorectal cancer with similar immune function decline,which mainly were the decline of cellular immune function. Part five:Effects of recombinant attenuated Salmonella harboring 4-1BB ligand gene on rat experimental colorectal cancerObjective To investigate the immune change of tumor-bearing rats and the feasibility of prevention and treatment of colorectal cancer induced by attenuated Salmonella typhimurium vaccine strain expressing rat 4-1BBL gene.Methods Rats were given weekly subcutaneous injections of DMH(20mg/kg) for 20 weeks.Fourteen weeks after first injection,rats were averagely and randomly divide into control group,SL3261, SL3261 + plasmid and SL3261 + recombined plasmid group,then fed with 2 ml PBS, SL3261,SL3261 + plasmid and SL3261 + recombined plasmid group,respectively 3 times at 2 weeks intervals.In 16 weeks blood samples were drawn from right atrial for phenotypic detection.In 21 weeks tumor-bearing rats were sacrificed and the number and size of colon tumors and metastasis were recorded for tumor incidence.The level of phenotype change in peripheral blood cells and spleen cells was determined by flow cytometry.The contentsof IFN-γin the supernatant of splenocytes stimulated by PHA for 72 hours were measured by ELISA.Primary and metastatic tumors were studied pathohistologically by HE,and report gene GFP and 4-1BB lingand expression were detected by Double-Immunofluorescence.Results 9.4±4.3 tumors were found in SL3261 + recombined plasmid group,which was significantly lower than the other group(P＜0.05),18.2±4.7 in PBS,12.8±4.5 in SL3261 and 12.2±4.4 in SL3261 + Plasmid(PBS was lightly higher than SL3261 and SL3261 + plasmid,but no significant statistical(P＞0.05).With double-immunofluorescence,there were GFP and more 4-1BBL expression on the rats,which were fed with SL3261+ recombined plasmids,SL3261+ plasmids can also detect GFP expression,but little 4-1BBL,while SL3261 only little 4-1BBL.The contents of IFN-γin the cultural supernatants from splenocytes stimulated by PHA of each group were detected by ELISA.The results of peripheral blood and splenocytes showed that CD3~+CD25~+ T cells in the group of SL3261+ recombined plasmid were obviously higher than that of other groups(P＜0.05).There were significantly more IFN-γproduced in the group with SL3261 + recombined plasmid than that of other groups (P＜0.05).However,there were no differences among other groups(P＞0.05).Conclusions Attenuated Salmonella carrying pIRES2-EGFP-4-1BBL plasmid can achieve tumor-bearing rats 4-1BBL gene transfection,in vivo 4-1BB ligand high expression can enhance anti-tumor immune and inhibit the growth of experimental colorectal cancer in rats.