Study On The Anti-tumor Effects Of The Sonicate Vaccine In Vitro And In Mice

Objective:1. To explore the cytotoxicity of the cytotoxic Tlymphocyte (CTL) induced by SW480 sonicate sensitized dendritic cells (DCs) on the colon cancer cell line SW480.2. To investigate the antitumor effects of the sonicate of FBL3 combined with low-dose cyclophosphamide(Cy).Methods:l.PBMCs were separated from the HLA-A*0201 donor and DCs were cultuireed with rhGM-CSF, rhIL-4 and rhTNF-a. The same donor's primary CTLs were stimulated by DCs loaded with SW480 sonicate. The cytotoxicity of CTLs on SW480 (HLA-A*0201 positive) and K562 (HLA-A*0201 negative) was determined by theMTT method.2.Male C57BL/6J mice,6 to 8 weeks old, were selected and divided into 4 groups (12 in every group):①PBS control group(untreated)②CTX group③FBL3 sonicate immunised group④CTX and sonicate immunised group. On the fourth day after injection of CTX, the antibodies of mice serum from eyeball blood were detected. Four mice were killed in every group. Their spleen cells were cultured for two days with FBL3 sonicated antigen, then cytotoxic activity of spleen cells to FBL3 were performed.At the same time the percentage of CD4+CD25+Tcells in CD4+Tcells were detected by flow cytometry. The other mice were injected subcutaneously with 5×106 FBL3 cells. Observe and record the growth rate of tumor.Results:1.The cytotoxicity of the CTLs on SW480 is stronger than that on K562(P<0.05).2. (1) The ELISA shows that the antibodies in blank group,PBS group and CTX group were not detected, but they were detected in sonicate group and combination group in which the antibody titers were more than 1:2560.(2) Flow cytometric analysis displayer that the percentage of CD4+CD25+Tcells in CD4+Tcells in CTX group was lower than the one in PBS group (p<0.05); The percentage in combination group was lower than the one in sonicate group (p<0.05); But the percentages between CTX group and combination and between PBS group and sonicate group didn't have differences (p>0.05). Flow cytometric analysis displayed that the percentages of T cells in lymphocytes didn't have difference between every two groups (p>0.05).(3) At the ratios of E(Effect) to T(Target) as 20:1,10:1,5:1,2.5:1, the killing rates of spleen cells in CTX group,sonicate group and combination group were higher than that in PBS group (p<0.05), but there weren't difference between CTX group and sonicate group (p>0.05).The rate in combination group was higher than the rates in other groups (p<0.05).(4) The mean formation times of tumors in four groups were (5.13±0.78)days, (6.75±0.97)days, (6.63±0.85)days,(8.88±1.45)days, respectively. By statistical analysis, the formation of tumor in CTX group,sonicate group and combination group was later than that in PBS group (p<0.05),but there wasn't difference between CTX group and sonicate group (p>0.05); The formation was latest in combination group compared other groups (p<0.05).By observation at the same times, the tumors in CTX group,sonicate group and combination group were smaller than the tumors in PBS group, but there werenot difference between CTX group and sonicate group; The tumors in combination group were smallest compared with other groups.Conclusion:The DC vaccine can stimulate specific CTLs which can trigger cytotoxic activity on the target cells and this cytotoxicity is related to MHC restriction.Sonicate can make mouse produce polyclonal antibody; CTX may reduce the number of Tregs; Sonicte and CTX can enhance the cytotoxity of spleen cells and delay the formation of tumor. Every dates of experiments suggest combination of sonicate and CTX has the better anti-tumor effect...