A Series Of Studies On Active Immune Response Induced By Ablation Combined With Perinatal Injection Of Immune Adjuvant In Mice

Part Ⅰ Experimental Study on Thermal Ablation of Transplanted Liver Cancer Subscutaneously at Different Tempretures in MiceObjective:To investigate the proper ablation temperature that can induce the maximum anti-tumor immunity by using different temperature saline ablation with subcutaneous liver cancer in mice via recording the survival time, tumor volume,the immune cell numbers and heat shock protein expression in lesion.Method:After the subcutaneous tumor model was established in mice,120 mice were randomly divided into 5 groups, each group had 24 mice. We started experiment when the tumor diameter about 6～8 mm length after 7 to 12 days. Each group were given intratumor injection of 0.15ml saline with different temperatures respectively (50 ℃,60 ℃,70 ℃,100 ℃). Survival time, tumor size, residual rate of tumor, lymphocyte number, and expression of HSP70 were measured in each group.Results:① Survival time were different in each group (P<0.05), but the mice in 50 ℃ group had the longest survival time. ② Compared with conrtol group, tumor volumes in each ablation were reduced significantly (P<0.05), but the 50 ℃ group was the only group without tumor continuing to grow up as compared with the volume before ablation. ③ Confirmed by HE staining, there were no tumor cell necrosis in the control group, residual rate was 100%; residual tumor cells were found at one specimen in 50 ℃ group, residual rate was 16.7%; residual tumor cells were found at 2 specimens in each of the other groups, residual rate was 33.3%. ④ CD4+ cells number in 50 ℃ group was significant higher than those in other groups (P<0.05). No significant difference of CD8+ cells number was show between 50 ℃ group and control group (P> 0.05), but CD8+ cells number in 50 ℃ group was significant higher than those in other ablation groups (P<0.05). CD4+/CD8+ratio increased markedly in 50 ℃ group. Compared with conrtol group, the labeling index of HSP70 in each ablation group increased significantly (P<0.05), and labeling index in 50 ℃ group was the highest.Conclusion:Compared with different saline ablation tempereture to subcutaneous liver cancer in mice, ablation with 50 ℃ saline may induce a more intensive antitumor immunity.Part II:The Study of the Antitumor Effect of Thermal AblationCombined with CpG ODN in Treatment of Mice Live CancerObjective:To investigate the therapeutic effect of thermal ablation combined with CpG ODN in treatment of rice liver cancer.Methods:After the subcutaneous tumor model was established in mice,64 mice were randomly divided into 4 groups. Except the control group, the others mice were treated at seventh day and fourteenth day.A half of all groups were injected equate tumor cells at thirtieth day after 1st therapy, tumor volume, survival times were recorded, lymphocyte number and expression of HSP70 in tumor were measured.Results: ① Mortality rate of experimental groups are lower than control group (P<0.05), no tumor exist in experimental groups. ② Compared with control group,tumor volumes in each experimental group were reduced significantly (P<0.05),but the 100 ℃ combined with CpG ODN group reduced most significantly. ③ CD4+ cells and CD8+ numbers in each experimental group are significantly higher than control group (P<0.05),and the number of 100℃ combined with CpG ODN group is much more than other groups. ④ Compared with control group, the labeling index of HSP70 in each experimental group increased significantly (P<0.05), and labeling index in 100 ℃ combined with CpG ODN group are more significant.Conclusion:CpG ODN can strengthen the immunity of rice with Hepal-6,CpG ODN combined with thermal ablation can effective cure rice liver,and can reduce recurrence effectively.PartⅢ:Study of the Antitumor Effect of Inject Intratumorally with MIP-3α Solution in Treatment of Mice Live CancerObjective:To investigate the possibility of attracting peripheral dendritic cells(DCs) to migrate to tumors and inducing specific immune response against mouse liver cancer by intratumoral injection of macrophage inflammatory protein-3α(MIP-3α).Methods:After subcutaneous liver cancer models were established, the mice were divided into 3 groups randomly. The mice in MIP-3α therapy group and PBS control group were injected intratumorally with MIP-3α solution or PBS respectively, while blank control group were not treated. The mice were sacrificed at day 20 after injection and the number of DCs, CD4+ and CD8+ cells in the tumor tissue were detected by immunohistochemistry. The number and phenotype of infiltrating DCs were analyzed by flow cytometry. Moreover, ten mice in each group were observed in order to draw the tumor growth curve and calculate the survival time.Result: ① The number of CD4+, CD8+ cells and DCs in MIP-3α therapy group were significantly higher than those in other two groups. ② The expression of CD80 and CD86 of dendritic cells in MIP-3α therapy group was were significantly higher than those in other two groups (P<0.05). ③ Tumor growth speed was significantly slower (P<0.001) and survival time was significantly longer (P<0.05) in MIP-3α therapy group than those in other two groups (P<0.05).ConcIusions:① Peripheral DCs can migrate into tumor site by intratumoral injection of MIP-3a, and DCs can capture and present tumor antigen, which can effectively induce specific anti-tumor immune response. ② MIP-3α may play a part in promoting maturation of DCs in the micro-environment of subcutaneous liver cancer model.