| Background and objiectiveAs traditional three methods, surgery, radiotherapy and chemotherapy have achieved good effects on the treatment of malignant tumors since last century. But these treatments are not effective on all tumors, and sometimes can cause serious side effects.Taking the treatment of acute myeloid leukemia for example, chemotherapy has achieved good results, but there are still 15%-20% patients who could not achieve complete remission (CR, Complete remission).Even in patients who achieved complete remission, there are still some side effects like chemotherapy-related death, and minimal residual disease causing relapse make retreatments more difficult.Therefore, methods that have little damagements and can effectively control tumor growing are the urgent needs for clinical cancer therapy. As tumor immunology and molecularbiology achieved rapid development and cross-penetration between themself, tumor immunotherapy, based on the principles of immunology and on the methods of immunological techniques, has been on the transition period from laboratory research to safe and effective clinical trial. As deeper understanding of body's specific immune responses against tumor, the escaping mechanisms aganist tumor immune and tumor micro-environment,the new strategies and new ideas of tumor immunotherapy have been further researched and expanded.Refractory or relapsed tumors are closely related to tumor cells escaping from immune attacks. The mechanisms that have been recognized are as follows:(1) deficiency or modulation of tumor antigens, making tumor cells escape from immune attack; (2) The decreasement or absence of MHCⅠmolecules on the surface of tumor cells downregulate NK cells' and other immune cells'killing effects on them, and this also associated with tumor metastasis; (3) tumor cells secret negative-regulating cytokines, such as TGF-β, IL-10, VEGF, etc, inhibiting T cell differentiation, promoting Th1-Th2 balance shifting to Th2, lowering T cell adhesion and expression of costimulatory molecules, inducing tolerance of tumor-specific CTL. The newest research shows that tumor cells secrete Sema3A inhibiting DCs from mature and therefore influcing their immune functions; (4) FasL that expressed on tumor cells can induce apoptosis of lymphocytes which expressed Fas, and downtegulation of Fas expression on tumor cells also make them escape from lymphocyte-inducing apoptosis; (5) adhesion molecules and costimulatory molecules on the surfaces of tumor cells are often defective, which influce the process of T cell being effectively activated due to lack of the second signals, and some negative costimulatory molecules such as B7-H4,are highly expressed; (6) tumor cells can induce the accumulation and amplification of a variety of immune-suppressing cells in the local area of tumor, which are closely related to tumor development. Those immune-suppressing cells that have been widely studied in recent years includes regulatory T cells (Treg), tumor-associated macrophages(TAM), myeloid derived suppressor cells (MDSC), etc; (7) tumor antigens can induce immune tolerance. Of course, the mechanisms of tumor cells escaping from immune attack are very complex, and many need be further researched,also several different mechanisms are often synergistic. Only deeper understanding of the mechanisms that how tumor cells evade immune attack, can the designment and implementation of specific immune treatments on tumors be made into breakthrough.The methods of immunotherapy which are designed according to the mechanisms that tumor cells escaping from immune attack, mainly include the specific active and passive immunotherapy. The former refers to the tumor vaccines, which can stimulates body to produce specific immune responses aganist tumor antigens, including tumor cell vaccines, tumor antigen vaccines, dendritic cells (DCs)-based vaccines and DNA vaccines.The latter refers to various monoclonal antibodies and adoptive immunotherapy.In essence, the main purpose of tumor vaccines is to screen specific tumor antigens that have strong immunogenicities and to enhance body's immune responses to them,then make tumor cells cann't escape from immune attack and be killed. So how to screen tumor specific antigens that have strong immunogenicities is the key of tumor vaccines designment, it is also the limit of tumor vaccines development.In our study, we use mouse mastocytoma P815 cells as antigens to immunize BALB /c mice subcutaneously.we want to investigate whether they can induce BALB/c mice to produce strong and specific immune responses against P815 cells. this may provide basis for us to screen antigens to design tumor vaccines.MethodsChapter one Establishment of BALB/c mice mastocytoma/mast cell leukemia modelsMouse mastocytoma P815 cells were injected into BALB/c mice through tail veins. The experiment was divided into seven groups, namely the group receiving 1×107,5×106,2.5×106,1×106,5×105,1×105,0 (RPM I 1640 medium as control group) P815 cells per mouse,six BALB/c mice for each group. Observed the survival state of mice,appearance and pathology of lung,spleen,liver, blood cells smear, bone marrow cells smear; measured the weight of body,lung,spleen and liver;did WBC and PLT counts.Chapter two P815 cells injected subcutaneously influence BALB/c mice forming mastocytoma/mast cell leukemiaThe experiment was divided into four groups, including P815 cells group, P815 cells that were dealed with mitoxantrone group, L1210 cells group and solvent control group, six BALB/c mice for each group.5×106P815 cells(PBS liquid preparation,0.5ml) per mouse,5×106 P815 cells that were dealed with 0.1ug/ml mitoxantrone for 12 hours (PBS liquid preparation,0.5ml) per mouse,5×106 L1210 cells (PBS liquid preparation, 0.5ml) per mouse and PBS liquid (0.5ml) per mouse were injected subcutaneously into the bilateral hips of each group mice respectively. A week later, mice of four groups were injected through tail veins with 1.5×106 P815 cells (RPMI1640 media preparation, 0.15ml) per mouse. Observed the survival time, blood cells smear, weight,lung,spleen and liver changes and evaluate the formation of mastocytoma/mast cell leukemia of all groups mice (observation deadline:three months after mice be injected with P815 cells through tail veins).Chapter three P815 cells injected subcutaneously induced BALB/c mice to produce specific anti-tumor immune responsesThe experiment was divided into two groups. Three BALB/c mice that were not dead after intravenous injection of P815 cells in the experiment of chapter two were included in the experimental group, newly purchased three BALB/c mice as control group. Control group mice, a week after injected with 0.5ml PBS liquid into bilateral hips, together with the experimental group mice, were killed by cervical dislocation. Removed the spleen sterilely and prepared the spleen cells suspension. Lactate dehydrogenase (LDH) release assays tested the killing rates of mice spleen cells from two groups when targeted on P815 cells and L1210 cells respectively. How did mice spleen cells from two group affect P815 cells colony formations were observed. each experiment repeated three times.The experimental datas of three Chapters indicated with mean±standard deviation (x±s). We used SPSS13.0 software for statistical analysis, body,lung,liver and spleen weights, white blood cells counts and platelets counts were analysed with one-way analysis of variance (One-Way ANOVA), when homogeneity of variances, we used LSD method for multiple comparisons, when heterogeneity of variances,we used Welch method that could correcte F values to test them and Dunnett T3 method for multiple comparisons. Kaplan-Meier survival analysis was used to analyze the survival time of mice. two group mice spleen cells'killing rates and colony affection on P815 cells, L1210 cells were analysed with Independent samples t test and two-factor two-level factorial design analysis of variance. P<0.05 indicated significant difference.RESULTSChapter one Establishment of BALB/c mice mastocytoma/mast cell leukemia modelsMice from groups that injected with P815 cells≥1×106 per mouse were all dead,< 1×106 per mouse all alive. We observed two mice which were injected with 1×106P815 cells got posterior limbs weakness between 12 days to 15 days after injection, then posterior limbs paralyzed and became stiff, and mice went to die later. Kaplan-Meier (Log rank) survival analysis revealed, there was a significant difference on overall survival time between death groups mice (mice injected with 1×107,5×106,2.5×106,1×106P815 cells) (x2=42.601, P<0.001); multiple comparisons also show significant differences (P<0.05). the median survival time of mice that injected with 1×107,5×106> 2.5×106,1×106P815 cells were 7.0days,9.5days,10.5days,17.5 days respectively,showed death groups mice injected with more P815 cells, the shorter survival time they got. compared to non-death groups mice (mice injected with 5×105,1×105,0P815 cells), the weights of death groups mice reduced (F=29.235, P<0.001),liver,lung and spleen weights significantly increased (F=46.939,14.940,31.995 respectively,P<0.001 all), WBC and PLT counts decreased (F=8.707,23.018 respectively, P<0.001 both);texture of livers became harder and a large number of white nodules in various sizes presented on their surface, hemorrhages could be seen in partial lungs and spleens, liver and spleen pathology showed a large number of abnormal-shaped cells in various sizes invaded them, a few mastocytoma cells could been seen on the blood cells smear, bone marrow cells smear observation showed the proportion of bone marrow blast cells became higher.Chapter two P815 cells injected subcutaneously influence BALB/c mice forming mastocytoma/mast cell leukemia.During three months of observation, there were four mice survived and two died on 14 days,17.5 days respectively after P815 cells were injected through tail veins in P815 cells group, there were five mice survived and one died on 17 days after P815 cells were injected through tail veins in P815 cells dealed with mitoxantrone group, mice from L1210 cells group and solvent control group all died between 12 days to 18.5 days after injected intravenously with P815 cells. Kaplan-Meier (Log rank) survival analysis revealed, there was a significant difference on overall survival time between all groups mice (x=16.585, P=0.001); multiple comparisons showed there were significant differences beteen P815 cells group mice,P815 cells dealed with mitoxantrone group mice and L1210 cells group mice,solvent control group mice(P<0.05), there were no significant differences beteen P815 cells group mice and P815 cells dealed with mitoxantrone group mice,beteen L1210 cells group mice and solvent control group mice(P>0.05).Compared to L1210 cells group mice,solvent control group mice, the weights of mice that were alive in P815 cells group,P815 cells dealed with mitoxantrone group significantly increased (F=41.467, P<0.001), liver,lung weights significantly reduced (F=13.283,5.658 respectively, P<0.000,=0.007 respectively), spleen weight are about the same (F=1.067, P=0.389);Blood cells smear and liver,lung,spleen pathology observition showed normal. The results of blood cells smear and pathology of liver,lung,spleen observition in dead mice from four groups were the same with that of mastocytoma/mast cell leukemia BALB/c mice,which demonstrated mice were all died from mastocytoma/mast cell leukemia.Chapter three P815 cells injected subcutaneously induced BALB/c mice to produce specific anti-tumor immune responseSpleen cells from the experimental group mice have no obvious morphological differences when compared to that of control group mice. spleen cells from the experimental group mice, either with 10:1 or 20:1 ratio targeting on P815 cells, the killing rates induced were significantly higher than that of spleen cells from control group mice(t=11.717,15.484 respectively, P<0.001 both),while there were no significant differences between spleen cells from two groups mice when either with 10:1 or 20:1 ratio targeting on L1210 cells (t=0.037,1.449 respectively, P=0.972,0.221 respectively).From this,we can see that spleen cells from the experimental group mice have specific cytotoxicity toward P815 cells. Compared to control group mice spleen cells, the abilities of prohibiting P815 cells colony formation from the experimental group mice spleen cells were significantly enhanced when either with20:1 or 50:1 ratio targeting on P815 cells (t=-11.538,-6.886 respectively, P<0.001 both).Conclusion1. By intravenous injection of P815 cells, BALB/c mice can get mastocytoma/mast cell leukemia. The main biological characteristics of this tumor model are as follows:①intravenous injection≥1×106 P815 cells per mouse can this model achieved, and when injected with more P815 cells, the shorter survival time mice got.②P815 cells maily invade liver, spleen and nervous system of mice,③after injection of P815 cells, mice peripheral white blood cells counts decreased slightly,shows it is a model of low proliferation of leukemia, platelets couts also decreased.a few P815 cells could been seen on the blood cells smear, bone marrow cells smear observation showed the proportion of bone marrow blast cells became higher than that of normal mice.2. BALB/c mice which were injected subcutaneously with either P815 cells or P815 cells dealed with mitoxantrone were both induced to produce a class of lymphocytes in spleen that have powerful and specific abilities to kill P815 cells and killed the follow-up intravenously invasive P815 cells, thereby they couldn't get mastocytoma/mast cell leukemia and survived. When subcutaneously injected with either L1210 cells or PBS liquid, BALB/c mice couldn't achieve such abilities,and went to die from mastocytoma/mast cell leukemia after subsequent intravenous injection of P815 cells. so the production of such killing ability was also specific.The innovation of our research1. we established an model of mastocytoma/mast cell leukemia on BALB/c mice and for the first time described the main biological characteristics of this model.2. we demonstrated that P815 cells which were injected subcutaneously could induce BALB/c mice to produce specific, strong immune response aganist P815 cells tumor for the first time.The value of our research1.By the establishment and main biological characteristics description of BALB/c mice mastocytoma/mast cell leukemia model,we provide a tumor model for researchers who engaged in tumor researches.2.P815 cells which were injected subcutaneously could induce BALB/c mice to produce specific, strong immune response aganist P815 cells tumor,which demonstrated that the immunogenicities of P815 cell autoantigens are very powerful.then we can screen antigens on P815 cells that have strong immunogenicities to design tumor vaccines.The shortage of our researchP815 cells origined from DBA/2 mice, which are allogeneic when compared to BALB/c mice. BALB/c mice can get tumor by intravenous injection of P815 cells,which suggests differences between their genes are very small, antigens origined from these different genes also induced mice to produce anti-tumor immune responses, but they are heterogeneous antigens to BALB/c mice, so they are meaningless for tumor vaccines research. If anti-tumor immune responses induced by those antigens are strong enough, then the research values of this experiment should be re-evaluated. |
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