| BackgroundDendritic cell is a kind of professional antigen presenting cell with the function of uptaking,processing,and presenting antigenic component for the other cells of the immune system.Due to the special features of the dendritic cells,the therapeutic tumor vaccine based on dendritic cells has been used to treat the neoplastic disease of human gradually.However,research shows that the immature dendritic cells can not present antigen effectively in the tumor environment.In order to overcome this limitation,the ex vivo culture of bone marrow cells are commonly performed.Following the acquisition of immature dendritic cells from the bone marrow cells,the tumor specific antigen will be administered to activate the immature dendritic cells so that these cells can become mature gradually by the stimulation of inflammatory stimulus.Currently,the dendritic cell based tumor vaccine immunotherapy has been used in numerous experimental immune treatment of human neoplastic disease,and the dendritic cells applied in these therapeutic tests were all from the patients themselves.For the dendritic cell based tumor vaccine,their mechanism of action is strongly associated with the clinical treatment of a variety of human malignancies,and this therapeutic method may be translated into clinical practice in the near future,which will bring a novel method for the systemic treatment of cancer patients.However,up to now,the relevant clinical trials have not shown favorable overall effectiveness in the treatment,the curative effect of dendritic cell tumor vaccine was confined to a small number of' patients.The migration of tumor vaccine to the secondary lymphoid organ or tissue will greatly affect the effectiveness of immune therapy since the premise of antitumor immune response activation is that the viable dendritic cell tumor vaccine could get to the secondary lymphoid organ or tissue successfully.The injection route and frequency of dendritic cell tumor vaccine along with the injected cell number are closely related to the in vivo migration efficiency of viable dendritic cells,and these factors will also determine the outcome of subsequent treatment.To date,there is still no standard method for the preparation and application of the dendritic cell tumor vaccine.Although relevant studies have demonstrated that ex vivo preparation of tumor specific antigen pulsed dendritic cell tumor vaccine is feasible and effective,the issue concerning the best injection route of tumor vaccine is still controversial.Given that a mass of draining lymph nodes as well as the spleen which is the largest lymphoid organ of the body exist in the abdominal cavity,theoretically speaking,intraperitoneal injection of dendritic cell tumor vaccine should be an effective method.However,till now,few relevant studies have been reported to support this point of view,thus the effectiveness and efficiency of this treatment method need to be further confirmed by animal study.In light of the research status described above,our study plans to explore a more efficient method for the acquisition and in vitro maturation of dendritic cells on the basis of the previous research,and to verify whether the intraperitoneal injection method could promote the migration of dendritic cells to the draining lymph nodes,lymphoid tissues,and lymphoid organs through the experiment in mice in vivo.In addition,our experiment also intends to study the effectiveness of intraperitoneal injection of dendritic cell tumor vaccine in the treatment of osteosarcoma through the mouse model of osteosarcoma,so that we can provide a new idea for the clinical treatment of osteosarcoma.Part 1Dendritic cell migration in abdominal lymph nodes-mechanism research of osteosarcoma immunotherapyPurposes:To explore a more efficient method for the acquisition and in vitro maturation of dendritic cells on the basis of the previous research,and to verify whether the intraperitoneal injection method could promote the absorption and migration of dendritic cells through the experiment in mouse model.Methods:Acquisition of bone marrow cells was achieved by flushing the marrow cavity of femur and tibia in C57BL/6 mice.The RPMI 1640 culture medium with 10%fetal bovine serum was used to culture mouse bone marrow cells,and the rm-GM-CSF(10 ng/ml),rm-IL-4(1 ng/ml),penicillin(100 units/ml),streptomycin(100 ?g/ml)and amphotericin B(0.25 ?g/ml)were added into the culture medium to induce the differentiation of dendritic cell precursors to immature dendritic cells.The IFN-?(100 ng/ml),LPS(250 ng/ml),and TNF-?(20 ng/ml)were added into the cell culture medium on the 7th day of cell culture so that the immature dendritic cells could be transformed into mature dendritic cells gradually,and the mature bone marrow derived dendritic cells were harvested on the 8th day.Then,the harvested bone marrow cells were tested by flow cytometry to find out the expression of CD 11c,CD11b,CD40,CD86,CD80,H-2Db,H-2Kb and MHC ?,which could be used as the molecular markers of purity and maturity for the dendritic cells.Through the intraperitoneal injection of india ink,the distribution of lymph nodes and lymphoid tissues of the mouse abdominal cavity and the india ink staining were observed.The mature dendritic cells which had been labeled by the red fluorescent dye(PKH26)were injected into the abdominal cavity of the mouse,then the spleen,pancreas and intestine were all harvested.Subsequently,the methods of paraffin section,frozen section,H&E staining,DAPI staining,light microscope observation and fluorescent microscope observation were performed to detect the dendritic cells in the tissues mentioned above.Results:1.For each IouseC the acquired bone marrow cell amount was 1.0-1.2×107;after 8 days' in vitro cell culture,the harvested mature dendritic cell amount was 1-2 x 108.Compared with the previous studies,the amount of bone marrow cells and mature dendritic cells in our study increased significantly.Besides,the dynamic development process of bone marrow derived cells which have the typical dendritic cell characteristics could be observed under the light microscope during the in vitro cell culture(from the I st day to the 7th day).2.The flow cytometry test showed that after 8 days' culture the bone marrow derived cells could express large amount of CD1 lc,CD1 lb,CD40,CD86,CD80,H-2Db,H-2Kb and MHC ?,which indicated that the dendritic cell purity was relatively high and the mature dendritic cells accounted for the vast majority of the bone marrow derived cells.3.Thirty minutes after the intraperitoneal injection of india ink,the lymph nodes and lymphatics in the mouse abdominal cavity could be distinguished directly by the naked eye.The H&E slides showed that the india ink were absorbed markedly by the abdominal lymphoid organs.4.Fluorescent microscope observation found that the mature dendritic cells labeled by red fluorescence consisted in the frozen sections of mouse spleen,pancreas and intestine.The dendritic cell percentage in the spleen was quantified by the ImageJ software,and the results revealed that there were no significant differences in dendritic cell percentage among different time points after the intraperitoneal injection.Conclusions:The in vitro dendritic cell culture method used by our experiment could acquire large amount of mature mouse bone marrow derived dendritic cells.Moreover,the mature dendritic cells administered by the intraperitoneal injection showed a favorable absorption efficiency and migration efficiency.Part 2The in vivo study of dendritic cell tumor vaccine immunotherapy of mouse osteosarcomaPurposes:To study whether the intraperitoneal injection of dendritic cell tumor vaccine could induce effective tumor specific cytotoxic T lymphocyte response in osteosarcoma mouse model,and whether this method could be an effective treatment for mouse osteosarcoma.Methods:The C3H mouse osteosarcoma model was established by the subcutaneous injection of LM8 osteosarcoma cell(2x106)in back,and the mice were divided into control group and experimental group randomly.The preparation and intraperitoneal injection method of dendritic cell tumor vaccine were as follows:The cell lysate of LM8 osteosarcoma was acquired by the ultraviolet B irradiation method.Then,the bone marrow cells of' C3H mice which had been cultured in vitro were co-cultured with tumor cell lysate to get the tumor antigen pulsed dendritic cell tumor vaccine.The pulsed dendritic cell tumor vaccine was injected into the abdominal cavity of the experimental group(1.5ml PBS containing 5x106 cells)on day 14,21,and 28,respectively.Meanwhile,the same volume of' saline was injected into the abdominal cavity of the control group.Each group was randomly divided into three subgroups according to the survival time(7 days,21 days,and 35 days).Each corresponding subgroup was sacrificed on day 7,21,and 35,and the tumor volume of each mouse was recorded.Furthermore,the spleens of healthy mice,tumor-bearing mice treated with mature dendritic cells which were not pulsed with tumor antigen,and tumor-bearing mice treated with pulsed dendritic cells were harvested.The CD8a positive T cells in spleen were isolated and purified by the CD8a positive T cell isolation kit and LS column.Then,the cytotoxicity test of the three groups of purified CD8 positive T cells was conducted by a standard lactate dehydrogenase cytotoxicity detection kit.Finally,the mouse osteosarcoma tissue slides of the control group and the treated group were prepared,the TUNEL staining was conducted,and the quantitative analysis of the images which were acquired from the stained slides was performed to test the apoptosis of the tumor cell.Results:1.The mean value and standard deviation of tumor volume were calculated for each subgroup of mice,and the tumor growth curves were drawn.Compared with the control group,the tumor growth of dendritic cell tumor vaccine treated group was significantly inhibited(P<0.0001).2.The cytotoxicity test found that T cells from the tumor-bearing mice treated with pulsed dendritic cells can generate stronger cytotoxicity than the naive T cells.Besides,T cells from the tumor-bearing mice treated with pulsed dendritic cells can also generate stronger cytotoxicity than the T cells from tumor-bearing mice treated with mature dendritic cells which were not pulsed with tumor antigen.As the T cell/tumor cell ratio increased gradually,the cytotoxicity of CD8 positive T cell also increased.The results of cytotoxicity tests were consistent with the results of therapeutic trials.3.The results of the TUNEL staining showed that the tumor cell apoptosis could be induced by the dendritic cell-based tumor vaccine in the treated group.According to the analysis results of the HistoQuest software,on the seventh day of the experiment,the apoptosis index of the two groups was at the same level,there was no significant statistical difference between the two groups(P=0.7650);on the twenty-first day of the experiment,the apoptosis index of the treated group was much higher than that of the control group,the difference between the two groups was statistically significant(P<0.0001);on the thirty-fifth day of the experiment,the apoptosis index of the treated group was also much higher than that of the control group,the difference between the two groups was also statistically significant(P<0.0001).Conclusions:For the antitumor immunotherapy,intraperitoneal injection of a massive dose of therapeutic dendritic cell tumor vaccine could generate effective tumor specific cytotoxic T lymphocyte response in mouse,and could effectively inhibit the growth of mouse osteosarcoma.Since this treatment method could be directly translated into the clinical treatment of human osteosarcoma,besides the surgical method and the neoadjuvant chemotherapy,it may become another effective treatment method for the human osteosarcoma. |
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