| Objective: We used small dose of cisplatin combined with DC vaccinesto target the the transplantable cervical carcinoma in mice. To observe theexpression of the immunosuppressive factors TGF-β1and IL-10in the tumortissue, and the infiltration of CD3~+T cells in the tumor tissue and CD4+,CD8+T cells in the spleens of the tumor-bearing mice. Explore whether the useof cisplatin can improve the anti-tumor effect of DC vaccine.Methods:1Setting up the tumor-bearing mouse model: Mice cervical cancer U14cells in the logarithmic growth phase were counted with a counting board andits concentration was adjusted to1×10~6/0.2ml for injection. The1×10~6(0.2ml)U14cells were then subcutaneously injected into the C57BL/6mice.Tumor-inhibiting experiment was conducted after7d being inoculated.2Preparing DC vaccine: Taking the U14cells in the logarithmic growthphase, which were inactivated by ray irradiation and preparing tumor antigenby repeated freezing and thawing. Mice were killed by cervical dislocationand bone marrow cells were got under aseptic conditions. The mouse bonemarrow-derived dendritic cells were induced and cultured by the rmGM-CSFand rmIL-4in vitro. Mouse bone marrow-derived DC and inactivated tumorantigen were mixed and cultured, that was DC vaccine pulsed with tumorantigen, which was identified by flow cytometry.3Tumor-inhibiting experiment: Success tumor-bearing mouse modelswould be divided into4groups with10in each: in control group, each mousewas injected intraperitoneally with0.2ml normal saline once per day for14days; in DC vaccine group, each mouse was given DC vaccine by local tumorinjection twice, an interval of one week; in cisplatin group,0.2ml cisplatin was given by intraperitoneal injection once per day for14days; in cisplatin andDC vaccine group, each mouse was given0.2ml cisplatin once per day for3days and then DC vaccine by local tumor injection, a week later, repeating theabove steps. The tumor growth curves were depicted, and the tumor wasweighed, with the tumor inhibition rate (IR).4Immunohistochemical SP method: The expression of TGF-β1andIL-10in the tumor tissue, and the infiltration of CD3~+T cells in the tumortissue and CD4~+, CD8~+T cells in the spleens of the tumor-bearing mice weredetected by immunohistochemical method. The Motic Med was used forquantitative analysis of the immunohistochemical results. The average opticaldensity values was used for the expression of TGF-β1and IL-10; percentageof positive cells was used for the percentage of CD3~+, CD4~+and CD8~+T cells.5Data analysis: Data analysis of variance of multi-sample meancomparison and S-N-K test was applied with SPSS13.0(size of test α=0.05).Results:1Identification of DC: DC had dendritic-like protrusions after culturedfor7days by inverted microscope, mostly suspended growth. The purity ofCD11c~+CD86~+DCs was89%by flow cytometry, that is typical and matureDCs.2Growth situation of tumor-bearing mice in each group: The growthstatus of mice in each group were all good1to10days after inoculation. Themice of control group began to decline in weight, appeared less activity andemaciation after15days. The mice of cisplatin group were in good conditionat the beginning of the medication, and then the feeding were significantlyreduced about a week after the medication; listlessness and slow activitiesabout two weeks after the medication. The mice of combined treatment groupand DC vaccine group were in good condition, the weight did not significantlydecline.3The impact of cisplatin and DC vaccine on transplanted tumor in mice:The tumor growth levels were basically same1to10days after inoculation,11to15days after inoculation, tumors in control group grew faster than other groups and tumors in combined treatment group grew slowest.16to24days,tumors in control group grew significantly faster than other groups, tumors incisplatin group and combined treatment group began to shrink. Tumors in DCvaccine group began to slowly shrink after18days, with the shrink being mostobvious in combined treatment group. The tumors weight in control group,DC vaccine group, cisplatin group and combined treatment group were(3.159±0.005)g,(2.794±0.006)g,(1.976±0.006)g and (1.736±0.005)grespectively. The tumors in every drug group were lighter than those in controlgroup (P<0.05); comparison among groups of the three drug groups were allstatistically different (P<0.05). According to the formula, the tumor inhibitionrate in DC vaccine group, cisplatin group and combined treatment group wererespectively11.55%,37.45%,45.08%. The combined treatment group had thehighest tumor inhibition rate, followed by cisplatin group and DC vaccinegroup; comparison among groups of the three drug groups were all statisticallydifferent (P<0.05).4Immunohistochemical results: The average optical density values ofTGF-β1in control group, DC vaccine group, cisplatin group and combinedtreatment group were (0.467±0.003),(0.466±0.003),(0.137±0.004) and(0.140±0.003) respectively; average optical density values of IL-10wererespectively (0.477±0.006),(0.474±0.004),(0.136±0.004) and (0.138±0.005);percentage of CD3+T cells were respectively (0.203±0.007),(0.357±0.005),(0.200±0.008) and (0.502±0.009); percentage of CD4~+T cells wererespectively (0.071±0.007),(0.305±0.004),(0.066±0.006) and (0.311±0.007);percentage of CD8~+T cells were respectively (0.162±0.003),(0.396±0.003),(0.159±0.005) and (0.398±0.005). The expression level of TGF-β1and IL-10were statistically different between cisplatin group and DC vaccine group(P<0.05), control group (P<0.05); the expression level of TGF-β1and IL-10were statistically different between combined treatment group and DC vaccinegroup (P<0.05), control group (P<0.05). No statistical difference (P>0.05)were found in the comparison between cisplatin group and combinedtreatment group; no statistical difference (P>0.05) were found in the comparison between DC vaccine group and control group. The amount ofCD3~+T cells were statistically different between combined treatment groupand DC vaccine group (P<0.05), cisplatin group (P<0.05), control group(P<0.05) respectively. The amount of CD3~+T cells were statistically differentbetween DC vaccine group and cisplatin group (P<0.05), control group(P<0.05) respectively. No statistical difference (P>0.05) were found in thecomparison between cisplatin group and control group. The amount of CD4~+and CD8~+T cells were statistically different between combined treatmentgroup and cisplatin group (P<0.05), control group (P<0.05); the amount ofCD4~+and CD8~+T cells were statistically different between DC vaccine groupand cisplatin group (P<0.05), control group (P<0.05). No statistical difference(P>0.05) were found in the comparison between DC vaccine group andcombined treatment group; no statistical difference (P>0.05) were found inthe comparison between. cisplatin group and control group.Conclusions:1In this study, tumors formed when the amount of injected U14cellswas up to1×10~6. Tumors seldom formed or do not formed when the amount ofcells was less than1×10~6.2Small dose of cisplatin in the tumor-bearing mice can downregulate theexpression of immunosuppressive cytokine TGF-β1and IL-10, and relieve theimmunosuppression in tumor microenvironment.3DC vaccine was injected into local tumor of tumor-bearing mice, whichcan improve the amount of CD3~+T cells in the tumor tissue. First with smalldose of cisplatin in tumor-bearing mice, and then injection of DC vaccine inthe local tumor can significantly improve the amount of CD3~+T cells in thetumor tissue, and also downregulate the expression of immunosuppressivecytokine TGF-β1and IL-10, thereby enhancing the sensitivity of the host onthe DC vaccine and initiating initiative anti-tumor immunity.4With DC vaccine in the tumor-bearing mice, it can upregulate theamount of CD4~+and CD8~+T cells in spleens of mice, improve anti-tumorimmune response capability of peripheral immune system and produce the good therapeutic effect. |
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