| The incidence rate and mortality of cancer gradually increase. It has benn more obvious for the past 10 years. Now caner is the leading and second deadly disease, respectively, in city and urban of China. The cancer brings a great threaten to human's health, as well as a tremendous burden on patients, families and society. Now the main treatment methods for cancer include surgery, chemotherapy, radiation and biological therapy and so on. Surgery also is the first approach to choose. However, more 70% patients with malignant tumor lose the opportunity of radical surgery because of the too big tumor burden, badly physical conditions or badly cardiopulmonary function. Thus, mini-harmful, local ablation-this therapeutic tool attract wide attentions. The therapeutic method gains good therapeutic effects in many patients during middle/advanced stages.With the proceeding of ablation, sequential therapy of remainder and sub-clinical tumor after main tumor ablated is paid attention generally. The clinical researches show injecting some drugs in remainder(for example low concentration acetic acid, low concentration sodium hydroxide, high concentration sodium chloride, alcohol, chemotherapeutics and so on) can enhance the therapeutic effects of local ablation effect. However, effect on local cellular immune function after microwave ablation/cryoablation and how to improve anti-tumor immunologic function are the emphasis of research on local ablation immunity recently.CpG ODN is artificial, strong immunologic agonist. It is the bigest immunologic feature that it can activiate DC, make DC proliferation, enhance its activiation on initial T cell. CpG ODN can induce many cytokines production,especial IL-12 and IFN-γ, regulate immune response prone to Th1 In addition, CpG ODN can induce polyclonal B cells to proliferate; also can activate NK cells. The activated NK cells can produce IFN-γ,TNF-αand so on. These cytokines can regulate immune function; CpG ODN can activate T cells, prime specific immune response to tumor. These are main mechanisms for playing anti-tumor roles.On the basis of above idea, we design this test that using microwave ablation/ cryoablation treats BALB/c mice subcutaneous transplantation tumors, then inject peritumorally TLR9 agonist-CpG ODN after ablation 1h. The therapeutic function and effect on immunologic function of mice with tumor will evaluated.Part 1 Effects of TLR9 agonist-CpG ODN on lympocytes activiation and surface molecules expressed on DC in miceObjective:To evaluate the effect CpG ODN on lymphocytes activiation and surface molecules expressed on DC in BALB/c mice.Method:Lymphocytes from mice spleens were separated and activiated by different concertrations CpG ODN. The rates of CD69+CD4+T cells,CD69+CD8+T cells and CD69+CD19+B cells from mice spleens were measured with flow cytometry. DC were separated from mice bone marrow, are stimulated with CpG ODN. The expression rates of CD80,CD86 and MHCⅡon DC were evaluated with flow cytometry.Results: 1. The different concertrations CpG ODN could activiate CD4+T cells, CD8+T cells and B cells from mice spleens. There was a significant increase in rates of CD69+CD4+T cells,CD69+CD8+T cells and CD69+CD19+B cells vs the control group(respectively, F=10.501, F=16.570, F=1564.965, all P<0.05).2. The expression rates of CD80,CD86 and MHCⅡon DC stimulated with CpG ODN were higher significantly than the control group's(respectively, F=59.680, P <0.001; F=47.233, P<0.001; F=18.674, P=0.003). There was no significant difference between the LPS group and the CpG ODN group(P>0.05).Part 2 Curative effect of microwave ablation/cryoablation combined with TLR9 agonist-CpG ODN on mice with CT26 tumorObjective:To evaluate therapeutic effect of microwave ablation/cryoablation combined with CpG ODN on mice with CT26 tumor.Method:Colon carcinoma model was established by subcutaneously inoculating CT26 cells into the right flank in Balb/c mice. The tumor-bearing mice randomly were divided into 6 groups:the group of PBS injected in peritumoral area(the control group), the group of CpG ODN injected in peritumoral area, the group of microwave ablation(MA), the group of microwave ablation combinated with CpG ODN, the group of cryoablation, the group of cryoablation combinated with CpG ODN. Variation of tumor volume in mice is observed. The median survival time was evaluated. The tumor-inhibition ratio was measured. The local recurrence rate of microwave ablation/cryoablation group and microwave ablation/cryoablation combined with CpG ODN group was measured. Mice in the microwave/cryoablation group and the two combination groups with their tumor regressed were rechallenged with CT26 cells.Results:1. The four treatments of MA, MA combinated with CpG ODN, cryoablation, cryoablation combinated with CpG ODN could significantly control tumor growth, could completely subcutaneously transplanted tumors. The tumor-inhibition ratio of the MA group, the MA combinated with CpG ODN group, the cryoablation group, the cryoablation combinated with CpG ODN group were 97.64%,97.84%.96.22% and 97.75%.The group of CpG ODN injected in peritumoral area has no effect on controlling tumor growth.2. The median survival time in the MA group, the MA combinated with CpG ODN group, the cryoablation group and the cryoablation combinated with CpG ODN group was significantly longer than the control group(the PBS group) (P<0.000). There was no significant difference between the CpG ODN group and the PBS group(P=0.756).3. The local recurrence rate in the MA group was 26.7%; however it was 6.7% in MA combinated with CpG ODN group. But there lied no significant difference in the MA group and the MA combinated with CpG ODN group(P=0.330). The local recurrence rate in the cryoablation group was 33.3%; however it was 6.7% in the cryoablation combinated with CpG ODN group. There was statistic different between the cryoablation group and the cryoablation combinated with CpG ODN group(P=0.002).4. In tumor rechallenged test, the ratio of forming tumor in the WA combinated with CpG ODN group was 25.0%, significantly lower than 75% in the WA group (P<0.05). However, there was no significant difference between the WA group and the WA combinated with CpG ODN group(P=0.132).The ratio of forming tumor in the cryoablation combinated with CpG ODN group was 12.5%(1/8), significantly lower than 75.0%(6/8) in the cryoablation group(P<0.05). There was significant difference between the cryoablation group and the cryoablation combinated with CpG ODN group(P=0.041). The ratio of forming tumor of the cryoablation combinated with CpG ODN group was significantly lower than the cryoablation group's.Part 3 The immunological effect of microwave ablation/cryoablation combined with TLR9 agonist-CpG ODN on mice with CT26 tumorObjective:To evaluate immunological effect of microwave ablation /cryoablation combined with CpG ODN on mice with CT26 tumor.Method:The mice treatment groups were as same as the mice treatment groups in Part 2. The mice were sacrificed at 21std after treatment. The CD3+CD4+T, CD3+CD8+T lmyphocytes in serum were counted with flow cytometry. Serum levels of interleukin(IL)-2, interleukin(IL)-12 and interferon-y(IFN-γ) were assayed by ELISA. The infiltrating CD3+Tcells,CD45RO+ cells and the relative expression of granzymeB and FasL on immunocytes in tumor tissue were examined by using immunohistochemical detection.Results:1.Comparision with the control group(the PBS group), not only in the WA group/cryoablation group, but also in the WA/cryoablation combined with CpG ODN group, no significant difference was found in the rates of CD3+T, CD3+CD4+T, CD3+CD8+T lymphocytes in the four groups mice serum (respectivly, F =0.768,P=0.588; F=2.447,P=0.057; F=2.040,P=0.136).However, the cryoablation group and the WA/cryoablation combined with CpG ODN group had effect on the CD3+CD4+T/CD3+CD8+T cells ratio. Comparision with the control group(the PBS group), the ratio of CD3+CD4+T/CD3+CD8+T cells in mice serum in the above-mentioned four groups significantly increased (respectively, P=0.019; P=0.007; P=0.002). Although the ratio of CD3+CD4+T/CD3+CD8+T cells in mice serum of the WA group also increased, the increase had no statistic significance (P=0.051). There was no significant difference in the ratio of CD3+CD4+T/ CD3+CD8+T cells between the CpG ODN group and the PBS group(P=0.921). 2. Treatment in not only the PBS group but also the CpG ODN group had no effect on the level of IL-2, IL-12 and IFN-γ(respectivly, P=0.581; P=0.445; P=0.799).Compared with the PBS group, the level of IL-2, IL-12 and IFN-γin the WA/cryoablation group and the WA/cryoablation combined with CpG ODN group had significant increases(all P>0.05), and the level of IL-2, IL-12 and IFN-γin the WA/cryoablation combined with CpG ODN group were significantly higher than the WA/cryoablation group.3. The infiltrating numbers of CD3+T cells in the WA/cryoablation group was significantly higher than that in the control group(the PBS group)(respectively, Pwa =0.028,Pcryo=0.007).But its in the WA/cryoablation group was significantly lower than that in the WA/cryoablation combined with CpG ODN group (all P<0.05). The infiltrating numbers of CD45RO positive cells in the WA/cryoablation combined with CpG ODN group was significantly higher than those in the other four groups (all P<0.05).And the infiltrating numbers of CD45RO positive cells in the other four groups had no significantly difference (P=0.099). Comparison with the WA /cryoablation group, the expression of granzymeB in the WA/cryoablation combined with CpG ODN group was significantly higher(all P<0.05). The expression of FasL in the cryoablation combined with CpG ODN group was significantly higher than that in the WA/cryoablation group(respectively, P=0.010; P=0.010).Conclusion:1. CpG ODN can activiate diverse immunocytes. CpG ODN can increase the expression of the costimulatory molecules CD80,CD86 and MHCⅡon DC from the mice bone marrow.2. Comparision with the control group (the PBS group), the median survival time of the WA/cryoablation group and the WA/cryoablation combined with CpG ODN group are significantly longer. There is no significant difference between the above-mentioned four groups.3. The local recurrence rate of cryoablation combined with CpG ODN group is significantly lower than that of the cryoablation group. Facing the same tumor cells rechallenging, the ratio of forming tumor in the cryoablation combinated with CpG ODN group is significantly lower than that in the cryoablation group. There is a kind of descendent tendency in local recurrence rate and the ratio of forming tumor facing the same tumor cell rechallenging in the WA combined with CpG ODN group. There are much more CD3+in local tumor tissue of the WA/cryoablation combined with CpG ODN group than that in the WA/cryoablation group. That may be connected with the deceased local recurrence rate of the WA/cryoablation combinated with CpG ODN group. More tumor specific memonic cells might explain the decreasing ratio of forming tumor facing the same tumor rechallenging. These show that local ablation combinated with CpG ODN can improve immune function of mice with tumor.4. The CD3+CD4+T/CD3+CD8+T cells ratio in mice serum in the WA /cryoablation group and the WA/cryoablation combined with CpG ODN group increases. However, there is no significant difference between the above-mentioned four groups. The cytokine level in mice serum in above-mentioned four groups also increases, and the level of IL-2,IL-12 and IFN-y in the WA/cryoablation combined with CpG ODN group is higher than the WA/cryoablation group. The increase of these cytokines level might be correlated with be strengthened immunocytes' activiation. |
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