| PartⅠ Spontaneous cell fusion between BMSCs and glioma cells in vitroBackground and objective :Bone Marrow Mesenchymal Stem Cells(BMSCs)was drawing more attention because of its self-renew ability and multilineage differentiation potential. Recently, evidence indicated that BMSCs have the ability to migrate to tumor sites and exert vital effects on tumor growth and progression through direct or indirect interaction with tumor cells. But whether BMSCs could fused with glioma cells to exert biological effect is unknown. It’s purposed to explore that cell fusion between human glioma cells and BMSCs in co-culture system, and detect the biological change of fusion cells.Methods:In the present study,human glioma cells SU3 cells were transfected with red fluorescent protein(SU3-RFP cells). BMSCs were isolated from transgenic nude mice, of which all nucleated cells express green fluorescent protein(GFP), and the immunophenotype and multilineage differentiation potential of BMSCs were confirmed. In a co-culture system of SU3-RFP and BMSC-GFP,RFP+/GFP+ cells were detected and isolated by dual colors using FACS. The expression of both glioma cell and mesenchymal markers were examined by RT-PCR and Western Blot. Biological characteristics was detected by CCK-8, colony forming efficiency in soft agar and wound scratch assays. The xenografl assay was used to evaluate the tumorigenicity of SU3-RFP and RFP+/GFP+ cells.Result:BMSCs expressed GFP protein and high levels of CD105,CD90 and CD44,but very low levels of CD45 and CD11 b. BMSCs have the ability to differentiate into osteoblasts and adipocytes in vitro. The results of CCK-8 test and cell colony formation assays revealed that the fusion cells grew faster and formed more colonies than parental cells. The wound scratch assays revealed that fusion cells own increased migration ability.RFP+/GFP+ cells and SU3-RFP were inoculated into BALB/C mice subcutaneously,tumorigenic rate in nude mice was 100%(5/5). The volume of xenografts generated by fusion cells and SU3-RFP is 779.6 ± 177.78 mm2 and 414.01 ± 105.04mm2, respectively.The weight of xenografts generated by fusion cells and SU3-RFP is 0.62 ± 0.14 g and 0.33± 0.08 g,respectively.Conclusion: Spontaneously-formed fusion cells between BMSCs and glioma cells were detected in vitro. Fusion cells perseve specific markers of parental cells, and showed enhanced proliferation and tumorigenic potential.Part Ⅱ In vivo and in vitro test of angiogenic effect of fusion cellsBackground and objective: Tumor neovascularization is vital for tumor gowth,affluent blood supply and active angiogenesis are malignant characteristics of glioma. In this part,we aimed to investigate whether fusion cells have the capability to participate in angiogenesis in vivo and in vitro. Meanwhile, we detected the expression of stemness gene of fusion cells and parental cells to explorer the possible mechanism.Methods:We cultivated SU3-RFP, BMSCs and fusion cells on Matrigel for 24 hours, and the process of morphological changes was observed. We also cultivated these cells in endothelial differentiation medium for 24 hours, cells was then detected for the expression of vascular endothelial cell markers,including CD31, CD34 and VE-Cadherin, respectively,by RT-PCR, Westearn Blot and immunohistochemistry. To detect the angiogenic effect of RFP+/GFP+ cells in vivo, RFP+/GFP+ cells and SU3-RFP was injected to the right caudate nucleus of BABL/C nude mice respectively. MVD were tested by immunohistochemistry(IHC) staining of CD31. Meanwhile, the proportion of CD133+ cells in fusion cells and glioma cells was assessed by flow cytometry. RT-PCR was also used to examine the expression of stem cell genes.Results:When cultured on Matrigel,fusion cells gradually formed tubular-like structures in vitro. 24 hours after cultivated in endothelial differenetiation medium, fusion cells showed elevated level of CD31,CD34,and VE-Cadherin(markers of VEC) compared with SU3-RFP and BMSCs. For angiogenic effect in vivo, tumor xenografts was tested by immunohistochemistry(IHC) staining of CD31, the MVD-CD31 in each group were19.67±1.8 vessels(RFP+/GFP+cells) and 7.16±0.54 vessels(SU3-RFP), respectively.MVD was significantly greater in RFP+/GFP+ cells xenograft tumor compared with that in SU3-RFP xenograft tumor. Meanwhile, the expression of stemness factors(Nanog, Oct4,Sox2) in fusion cells increased, and the proportion of CD133+ cells increased in fusion cells compared with the parental cells by FACS.Conclusion: Fusion cells exhibited enhanced angiogenic effect compared with parental glioma cells in vivo and in vitro, which may relate to the stemness property.Part Ⅲ Study on the role of cell fusion in glioma neovascularization by dual-color orthotopic model of transplantable gliomaBackground and objective:In vivo studies of tumor neovascularization requires a better tracing model to show the relationship between tumor cells and the host. We intend to analysis tumor vascular structures using RFP/GFP dual-color fluorescence glioma model in nude mice, searching evidence of cell fusion involved in glioma neovascularization.Methods:SU3-RFP cells were injected into the right caudate nucleus of NC-C57Bl/6JGFP nude mice. After 4 weeks, transplanted tumor were sampled, part of the fresh tumor tissue was cut into 1 mm3 pieces and pressed on slides for fluorescence microscopy. Part of tumor sample was embedded in Optimal Cutting Temperature medium, frozen in liquid nitrogen. The frozen samples were continuously sectioned at a thickness of 5μm,then either observed under a confocal laser scanning microscope or performed immunohistochemical staining.Results:Under fluorescence microscopy, tumor(red) and peritumoral brain tissues(green)could be easily distinguished. A fraction of tube-like structures co-expressing RFP and GFP were detected in the preformed tissue slice suggesting the occurrence of cell fusion in the tumors.Transverse sections of the blood vessels were observed under confocal laser scanning microscope, RFP+/GFP+ cells was detected,immunohistochemical staining for CD105 and CD90 on adjacent section of the RFP+/GFP+ vessels was positive indicating that the RFP+/GFP+/CD105+/CD90+ cells on vessel wall originated from the fusion of SU3-RFP cells and host BMSCs.Conclusion:Dual-color fluorescent tracer model may play a unique role in distinguishing tumor vessels generated from tumor cells, host cells, or fusion cells in the study. It suggested that cell fusion was an important way of tumor neovascularization... |
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