| Current methods for cancer treatment are maily surgery, chemotherapy, radiotherapy and emerging biological therapy. Biological therapy is currently a hot topic and has a good prospects in cancer treatment field while Dendritic cells (DCs) is the most important focus in biological therapy. Dendritic cells are the most potent antigen presenting cell(APC) currently konwn and play important role in caner immunology. DCs vaccines are in an attempt to be used as therapeutic anticancer vaccines after loaded with tumor-associated antigens (TAAs), until now there are two methods to load the DCs with TAAs. The first one is that DCs loaded with lytic-Ag generated from cancer cells, the second is that DCs merged with cancer cells to acquire the cancer cells Ag. In our papers we used the second method to produce DCs vaccines, and this method is more direct and simpler than others.Exosomes are30-120nm membrane vesicles that are secreted into the extracellular milieu as a consequence of multivesicular body fusion with the plasma membrane. Many cells such as DCs, tumor cells, leukomonocytes, blood plates, mast cells, enterocytes, intestinal epithelial cells, schwann cells, fat cells, nerve cells, fibroblasts, and many tumor cell lines can release exosomes. At present, there are two types exosomes, one is TEX(tumor derived exosomes). It contains tumor antigens, so it can strengthen immunogenicity of tumor cells to promote antitumor immune responses. The other is DEX(dendritic cells derived exosomes). While some studies have also shown that exosomes derived from certain tumor cells and immature dendritic cells can not effectively enhance the body immunity, and even has a role in immune tolerance. Thus, the anti-tumor effects of exosomes are characterized by not only the source of the cells and their functional status, but also on the need of functional verification of exosomes in vitro or in vivo.Taking into account the antigen-presenting ability of DCs and the easy proliferation ability of tumor cells, some researchers made tumor vaccines by fusing myeloma cells with DCs using hybridoma technology. This method not only overcame the difficult problem of DCs separation, extraction and cultivation but also overcame the weaknesses of immunogenicity and poor antigen-presenting ability of tumor cells. Tumor vaccines have shown anti-tumor effects in some studies. But until now there have few reports on the immunological effects of the exosomes derived from these fusion cells. In this study, with the help of hybridoma technology, we fused myeloma cells and DCs, then extract exosomes from the fusion cells and to study their anti-tumor effect.ObjectIn order to get tumor therapy vaccines which are more stable, resist to high temperature compared to cell vaccines and can be tested in terms of doses, we extracted the supernatant of DCs/tumor cell fusion cells to isolated exosomes, then to identify them and to do study on their anti-tumor effects. Thereby we could open up new avenues for the applications of DCs/tumor cell fusion vaccine.Methods1. Extraction and cultivation of DCs:Bone marrow mesenchymal stem cells were separated and extracted form the femur, tibia and fibula of6-8weeks from BALB/c male mice. Bone marrow mesenchymal stem cells were rinsed and let stand for24h, then non-adherent cells were wiped off. The addherent cells were cultured in RPMI1640medium in a humidified atmosphere of5%CO2at37℃. The medium was supplemented with20ng/ml human recombinant GM-CSF, and10ng/ml human recombinant IL-4. The addherent cells were fed every2days by gentle replacement of50%of the medium with fresh medium and were induced to differentiate into DCs.2. Cultivation of myeloma sp2/0cells:Myeloma sp2/0cells were removed from the liquid nitrogen, after resuscitation myeloma sp2/0cells were cultured in DMEM medium with10%FBS. Myeloma sp2/0cells were treated with8-azaguanine(8-AG) two weeks before fusing with DCs.3. Fusion of DCs with sp2/0cells and identification of DCs with sp2/0fusion cells:Polyethylene glycol (Polyethylene Glycol, PEG) chemical fusion method and electric fusion method were conbined to prepare DC with sp2/0fusion cells. Using are combined preparation DC/sp2/0fusion cells. Fusion efficiency was determined by flow cytometry assay and fusion cells were identified by confocal laser scanning microscopy.4. Cultivation of DC with sp2/0fusion cells:DC with sp2/0fusion cells were cultured in96-well plates using DMEM selection medium which contained1%HAT(H-Hypoxanthine, A-Aminopterin, T-Thymidine) and10%FBS for14days. The fusion cells were then cultured using DMEM selection medium which contained1%HT (H-Hypoxanthine, T-Thymidine) and10%FBS for7days. After selection, fusion cells were cultured in DMEM medium with10%FBS and100ng/mlGM-CSF.5. Extraction and identification of exosomes:Supernatant of fusion cells were collected and exosomes were extracted by density gradient centrifugation methods. Microplate reader was used to measure the quantity of exosomes, transmission electron microscope was used to observe the morphology of exosomes and SDS-PAGE and western blot were used to identify exosomes.6. Extraction and cultivation of T cells:Mononuclear cells were isolated from6-8weeks BALB/c male mice spleen, then T cells were obtained by using nylon hair assay. The seperated T cells were cultured in DMEM medium with10%FBS supplemented with30p.g/ml IL-2.7. T-cell proliferation assays:T-cells were incubated with exosomes and MTT assay were used to check the influence of exosomes on the proliferation of T cells.8. Cytotoxicity assay of T cells after treated with exosomes derived from DCs, immuture DCs and fusion cells:T cells were incubated seperately with DCex^imDCex and exosomes for6days. Then LDH methods were used to check the cytotoxicity ability of T cells on cancer cells, T cells and Myeloma sp2/0cells were used as control.9.The result is analysed by the statiscal software SPSS13.0 Results1. Fusion and identification of DC with sp2/0fusion cells:7days after induction by GM-CSF、IL-4, adherent bone marrow mesenchymal stem cells could transform into DCs with typical morphology. The fusion efficiency were about45%checked by flow cytometry assay and double-positive fusion cells(MHC-Ⅰ、 MHC-Ⅱ positive) were observed under confocal laser scanning microscopy.2. Seperation and identification of exosomes derived from DC with sp2/0fusion cells:DC with sp2/0fusion cells were adherent growth with round morphology. Exosomes were seperated from the supernant of fusion cells and the exosomes expressed MHC-Ⅰ、MHC-Ⅱ moleculars at the same time identified by western blot assay.And control group sp2/0cells secreted exosomes without MHC-Ⅱ molecules3. Stimulation of T cells proliferation:Exosomes secreted by the fusion cells or matured DCs significantly promoted T cells proliferation, and the proliferation effects were positively correlated with the dose of exosomes. It was statistically significant compared with that in control groups(P<0.01). The exosomes secreted from immatured DCs could not promote T cells proliferation, and it had no significant difference compared with that in control groups(P>0.01).4. Cytolytic activity of the T cells:Exosomes secreted by the fusion cells or imDC could induce cytolytic activity of the T cells compared with that in control groups, and it was statistically significant (P<0.01). The cytolytic activity of the T cells was strongest when T-cells to tumor cells was at the ratio of50:1. While exosomes secreted from immatured DCs could not induce cytolytic activity of the T cells and it had no significant difference compared with that in control groups(P>0.05).ConclusionDC with sp2/0fusion cells had the ability to secret exosomes and the exosomes secreted from these fusion cells could significantly promote proliferation ability and cytolytic activity of T cells... |
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