| Part Ⅰ.Cells Identification and Compasion of Glioma-derived Mesenchymal Stem Cell and Bone Marrow-derived Mesenchymal stem cellBackground: Glioma related mesenchymal stem cells have been successfully isolated.And bone marrow mesenchymal stem cells have been shown to interact with glioma to promote angiogenesis and vascular structure.However,the characteristics,functions and interactions between glioma and gb-MSCs are remain elusive.Methods: gb-MSCs were isolated from glioma(who grade III and IV),then cultured with DMEM medium containing 10% fetal bovine serum at 37℃ contain 5% CO2.Bone marrow mesenchymal stem cells(bm-MSCs)were isolated from bone marrow.then cultured with DMEM medium containing 10% fetal bovine serum at 37℃ contain 5% CO2.To observe the morphology and growth feature of mesenchymal stem cells and bone marrow mesenchymal stem cells.The proliferation of gb-MSCs and bm-MSCs were detected by CCK-8 assay.Flow cytology was used to detect the expression of CD105,CD44,CD73,CD90,CD14,CD14,CD31,and PDGFR-β on gb-MSCs and bm-MSCs.the differentiation potential of gb-MSCs and bm-MSCs were evaluated by Oil Red O staining,Alizarin Red staining and Alcian blue staining.Results: Both the gb-MSCs and bm-MSCs were fusiform and adhered to the wall.Gb-MSCs and bm-MSCs were positive for CD105,CD44,CD90,and CD73,and negative for CD34,CD14,and CD31.But the expression of CD90,CD73 on gb-MSCs was significantly lower than bm-MSCs,moreover no PDGFR-β expression was found on gb-MSCs.Both the mesenchymal stem cells and bone marrow mesenchymal stem cells could differentiate into adipocytes,osteoblast and chondrocytes.Conclusions: MSCs could be isolated from glioma.The expression of CD90,CD73 and PDGFR-β were significant difference between gb-MSCs and bm-MSCs.These results suggest that gb-MSCs were not all recruited from bone marrow,and may play a more important role in glioma.Part Ⅱ.Glioma-dependent Differentiation and Angiogenic Capacity of Glioma-derived Mesenchymal Stem CellObjective: To explore weather glioma-conditioned medium can induce gb-MSCs differentiated into pericyte,and,observe the effect of glioma-conditioned medium on the proliferation and migration and angiogenic capacity of gb-MSCs.Methods: gb-MSCs and HUVECs were kept in Dulbecco’s modified Eagle’s medium(DMEM)and RPMI1640 supplemented with 10% fetal bovine serum respectively.Gb-MSCs were co-cultured with serum free DMEM(0%DMEM),DMEM contain 10% fetal bovine serum(10%DMEM),serum free glioma comdition medium(0%gb-CM)and standard glioma condition medium(S-gb-CM)respectively.gb-MSC to pericyte transition was detected by immunofluorescence staining and western blot assay;angiogenetic capacity was analyzed by tube formation assay.Results: glioblastoma-conditioned medium increased gb-MSC proliferation and migration capacity and is capable of inducing gb-MSC differentiation into pericytes.gb-MSCs incubated in glioma-conditioned medium formed more tube-like structures.glioma-conditioned medium could enhance the stability of blood vessels and promote the attachment of gb-MSCs to human umbilical vein endothelial cells(HUVECs)on Matrigel in vitro.Conclusions: glioma-conditioned medium can induce gb-MSCs differentiation into pericyte and promote the proliferation,migration and tube formation of gb-MSC.Part Ⅲ.Mechanism of Gb-MSC to Pericytes DifferentiationObjective: To explore the role of glioma-secreted pro-vascular factors in gb-MSC to pericyte differentiation,and observe the miRNA modification of gb-MSCs in glioma-conditioned medium.Methods: The level of VEGF、PDGF-β、FGF2、TGF-β in different condition medium were analyzed by ELISA assay.The α-SMA expression in 0%DMEM with or without VEGF were analyzed by immunofluorescence.The level of α-SMA protein in 0%gb-CM with or without anti-VEGF antibody were analyzed by westen-blot assay.The tube formation assay was used to detect the angiogenic capacity of gb-MSCs in 0%gb-CM with or without anti-VEGF antibody.Additionally,RNA was isolated from gb-MSCs,and miRNA modifications were analyzed using the RAffymetrix miRNA microarray.Results: The level of VEGF,PGDF-β,FGF2,and TGF-β were significantly difference between the malignant glioma-conditioned medium(gb-CM,0%gb-CM and S-gb-CM)and normal medium(0%DMEM and 10%DMEM)of malignant glioma,and the level of VEGF in gb-CM was significant higher than in normal mediumwas.Moreover,we found that VEGF can induce gb-MSCs differentiation into pericyte.In addition,the results showed that miRNA expression on gb-MSCs cultured in 0%gb-CM was significant different from cells cultured in 0%DMEM,and its signaling pathways is partially correlate to tube formation.Conclusions: Malignant glioma could secrete a variety of angiogenic factors,and VEGF play a important role in gb-MSC to pericyte differentiation.moreover,the Malignant glioma-conditioned medium can induce miRNA modification of gb-MSC. |
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