| Background and ObjectiveChemotherapy, surgery, radiotherapy are the three principal treatment of cancer. With the progress of the study, biological treatment of cancer plays increasingly role in cancer therapy, that has become the fourth curing method. Loss of expression of tumor antigens and Immune tolerance Important reason why body can not identified and attack the tumor. Recently, phenomenon and concept of "Immunogenicity of death" has been discovered, we have a new understanding of surface antigen expression in tumor and identification of the body of the tumor.Previous theory showed:Apoptosis is a non-immunogenic or immune tolerance. The necrosis is caused by a strong immune response. However, the above inference is inconsistent with the experimental results. Although from the perspective of morphology, apoptosis is the physiological process of homogenization. However, apoptosis is not a physiological process of homogenization analyzed from the change of surface molecules expression during apoptosis.Cell death includes apoptosis, autophagy, necrosis and mitotic failure, apoptosis and necrosis are common. Recent studies show that apoptosis can be Classified as physiological cell death and immunogenicity of death based on whether the surface expression of apoptosis caused by immune attack of the protein molecules. Anthracycline chemotherapy drugs enhance the immunogenicity of tumor cells which were apoptosising by promoting apoptosis of tumor cells exposed to the surface markers, releasing of substances out of the cell, changing the micro-environment-mediated, at the same time it induce tumor cell apoptosis.Such as promotes expression of cell membrane calcium-binding protein (calreticulin, CRT), heat shock protein (heat shock proteins, HSPs), MHC I molecules and promote the release of the immune stimulus (such as the secretion of high mobility group protein B1 (high mobility group box protein 1, HMGB-1), nucleic acid and its metabolites, etc.) and the change in the tumor micro-environment.Immune cells uptook these apoptotic cells, particularly by DCs, what induced the immune system to attack tumor cells. Chemotherapy not only had direct toxic effect on tumor cells toxicity but also Induced tumor immunogenicity. How to integrate collaborative means to improve a variety of anti-tumor efficacy of Cancer will be Important issue of cancer therapy in future. Make good use of the death of tumor immunogenicity of the molecular signature of biological therapy for cancer has great far-reaching significance. That helps to explore the combination of traditional chemotherapy cancer immunotherapy strategy and guide the efficacy of chemotherapy prognosis.Currently, studies about chemotherapy drug-induced blood tumor cell immunogenicity of the death are limited, especially there is no relevant reports in terms of establishing mice model. Our previous work has been successful in transplanting mice erythroleukemia (MEL) cells to the bodies of Kunming (KM) mice which have normal immune function to construct suitable mouse erythroleukemia model for this study. On this basis, selected anthracycline chemotherapy drug doxorubicin (adriamycin, ADM) role in MEL cells.MethodPartⅠTo determine the objective concentration target of ADMLogarithmic growth phase of the MEL cells were seeded in 6-well plates, (1×106 cells/well),990μL/well Cell suspension. ADM removed from each of the mother liquor, after dilution by adding 10μL cell suspension per well to a final concentration of ADM 1μg/mL,2μg/mL,4μg/mL,8μg/mL. Repeat for each concentration of 6 holes. Cells were collected to 1.5mlEP tube after 24h incubation to finish following two operations.①Estimates of different concentrations of ADM is a rough MEL proportion of apoptotic cells by Hoechst 33258 staining of apoptotic MEL cells;②Determined target concentration of ADM which affected apoptosis of MEL cells at DAPI-/AnneV+ and DAPI+/AnneV+ ratio and 70% of by flow cytometry. The experiment was repeated three times, each time a negative control are located.PartⅡMouse model of leukemiaMEL cells in logarithmic growth phase were collected in the centrifuge tube by centrifugation at 1000RPM for 5min. Washed 3 times with PBS. Resuspended in PBS to adjust the cell density was 5×10 cells/mL. Male KM mice were inoculated via the tail vein 1×106个(0.2mL) MEL cells and were observed. (1) Peripheral blood (PB):mice dying or 80d peripheral blood count WBC tail, Specimens from peripheral blood smear by Wright-Giemsa staining to observe Cell morphology and counted the percentage of nucleated cells under light microscope. (2) Bone marrow (BM): detected bone marrow blast cell percentage of mice immediately after Dying mice were killed by cervical dislocation. Produced bone marrow smears by Wright-Giemsa staining to count 500 nucleated cells and category count the cells under light microscope. (3) Histopathological:The liver, spleen and bone marrow of mice were made of paraffin sections when mice were killed to be dying. HE staining and observed under light microscope to determine the cause of death in mice.Part III Experimental study of pre-infusion mice erythroleukemia (MEL) cells of inducted by adriamycin to erythroleukemia mice by intravenous to extend the survival time30 male KM mice were randomly divided into murine erythroleukemia model group (model group), ADM induced treatment group (ADM group), blank control group (control group),10 mice each group. Model group(A):Intravenous injection of sterile PBS,200μL/each mouse.8 days later, Logarithmic phase of collecting MEL cell were injected to mice through tail vein injection,1×10 cells/each (200μL). ADM group (B):MEL cells were incubated in culture flasks with 4μg/mL ADM (Target concentration of ADM Determined by flow cytometry)for 24 h, harvested by centrifugation. After adjusted the cell density to 9×106 cells/each mouse(200μL), injected into mice by Intravenous injection.8 days later, dealt with the same as group A. The control group (C):Without any human intervention. We observed in mice every 1-2 d. Detected the total number of peripheral blood leukocyte (WBC) and body weight in 80 days or survival mice dying, and recorded prevalence and survival time in mice.Main outcome measures:①peripheral WBC count in 80 days or survival mice dying;②Weight of 80 days or survival mice dying;③Survival time of mice④PB smears and bone marrow (BM) morphology of smear in 80 days or survival mice dying;⑤Histopathological examination in 80 days or survival mice dying.Statistical MethodsStatistical data was statistical analyzed using SPSS13.0. Experimental measurement data was present as mean±standard deviation (x±s). Comparing mean of two groups was using two independent samples t-test, multiple groups were compared with one-way classification (One—way ANOVA), pairwise comparied between groups by LSD t test, when heterogeneity of variance pairwise comparied between groups by Dunnett,s T3. detected homogeneity of variance by Levene test. P <0.05 was statistically significant. Survival time using Kaplan-Meier survival analysis method.ResultPartⅠThe target concentration of ADMApoptosis result:DAPI-/AnneV+and DAPI+/AnneV+ratio is about 70% of the ADM when the concentration of 4μg/mL.PartⅡMouse model of leukemiaKM mice are 100% access to disease,they emaciation, arched and lazy move, eat less, dull coat, claudication, their liver and spleen was swollen. Dying mice was significantly higher WBC count. Dying mouse immature red blood cells in peripheral blood smear 3%-6% to middle and late erythroblast mainly seen the original and early erythroblastic. Nucleated cells in bone marrow smears showed marked hyperplasia of active proliferation of erythroid and myeloid obvious; erythroblastic≥50%, the original and early erythroblastic mainly seen dual-core. NEC in the original cell≥30%, myeloblast cells were round, fine chromatin, prominent nucleoli, little cytoplasm. Meet the diagnostic criteria for acute leukemia.Take incidence in the liver, spleen, bone marrow pathological examination, microscopy of liver, spleen damage to normal tissue structures, there is diffuse infiltration of leukemic cells; high dose group in the heart, kidneys can be found in the tumor cell invasion; vascular A tumor cell stasis. Morphology of tumor cells in tissue culture flask in MEL cells than change. Leukemia cells in bone marrow infiltration, the original and immature red blood cells, granulocytes was significantly increased.PartⅢExperimental study of pre-infusion mice erythroleukemia (MEL) cells of inducted by adriamycin to erythroleukemia mice by intravenous to extend the survival timeTo 80d, the model group (A) mice were all dead, dying mice appeared anorexia, lazy move, thin, curled up and so on; ADM group (B) there is still a mouse survival; the control group (C) small Rats all survived. A, B, C group when the mice dying or 80d:①the number of peripheral WBC were:16.06±3.38,11.02±2.69,5.81±0.88;②weight were:28.42±4.33,40.24±3.22,45.91±2.14;③survival time was:28.00±8.43,56.00±12.68,80.00±0.00. A, B 80d mice dying or the number of peripheral blood WBC, body weight and survival time were higher than the C group were lower, shorter. However, B A group of mice compared with mice in terms of:the number of peripheral WBC decreased (P <0.01), weight gain (P<0.01), prolonged survival (P<0.01).Conclusion1. Established erythroleukemia mouse model by use MEL cell infusion KM mice.They have the similar pathophysiology of human leukemia.2. driamycin of 4μg/mL effect on MEL cells after 24 h, flow cytometry DAPI-/AnneV+ and DAPI+/AnneV+ ratio about 70%, indicating that MEL cells induced apoptosis of early and late in the proportion about 70%,which can occur immunogenicity.3.Compared with the erythroleukemia mouse model group,the treated group were significantly reduced in the peripheral blood levels, body weight, survival time.The innovation of this study:Established the acute erythroleukemia mouse model by use MEL cell infusion KM mice; Measured concentration and time, and the phenomenon of animal experiments of the adriamycin which can induce the immunogenicity death of MEL cell.The value of this study:Explore the strategy of conventional chemotherapy combination immunotherapy, Guide the prognosis of chemotherapy. |
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