Study The Mechanism Of Mesenchymal Stem Cells Migrating Toward Glioma

Objective:To isolate and culture Mesenchymal stem cells(MSCs) from bone marrow,purify and identify them to obtain available MSCs strain.To investigate the capacity of MSCs migrate toward glioma,glioma's impact on MSCs' phenotype,the effect of PDGF on the ability of MSCs migrate to glioma.Methods:(1) isolate and culture MSCs by Whole bone marrow culture method. Inverted microscope to observe cell morphology and cell arrangement,Flow cytometry to identify cell surface markers of MSCs,depict cell growth curve by MTT method.(2) The chemokinetic activity of MSCs in response to glioma cells Was evaluated by Transwell chemotaxis model.(3) Rat MSCs were co-cultured with glioma cells or induecd by condition medium,Use inverted microscope to observe the change of cell morphology after aboves culture,Immunofluorescent staining of above cultured cells forⅧand KDR.(4) The expression of PDGFR-αand PDGFR-βon human MSCs were evaluated by RT-PCR. (5)Use in vitro Transwell chemotaxis model,to investigate if PDGF can attract MSCs targeted transfer,different concentrations of PDGF on the ability of MSCs' directional transfer,Evaluate the effect of PDGF on MSCS directionally migrate to glioma.Results(1) The MSCs have been successfully isolated from bone marrow.Long spindle-shaped cells,arried in Whirlpool with typical,Rat MSCs cell surface markers:CD90,CD105 positive expression,Human MSCs cell surface markers:CD44,CD90 positive expression,CD34,CD45 expression was negative.MTT assay results showed that different generations of cells have the similar proliferation characteristics,Day three after passage to enter the exponential growth phase,With the passage there is attenuation trend of cell growth.(2) In vitro Transwell chemotactic migration model confirmed that glioma cell cultured supernatant can attract MSCs targeted transfer.(3) immunofluorescent staining showed that co-culture with glioma cells or cultured by glioma-conditioned medium,MSCs expression of vascular endothelial cell markerⅧand KDR.(4) The results of RT-PCR showed that human MSCs expressed PDGFR-αand PDGFR-β.(5) In vitro Chemotactic migration experiments showed that the transfer cells attracted by different concentrations of PDGF were significantly different from the control group,and the higher the concentration,the cell transfer more(P＜0.05),condition medium of U87 glioma cells can attract MSCs trageted migration,the number of transfer cells are more than the control group,there is significant difference,after adding PDGF antibody in the condition medium of U87 glioma cells,the number of targeted migrated MSCs significantly reduced(P＜0.05),But still more than the control group.Conclusion(1) through the whole culture of bone marrow cells can obtain purified MSCs lines,and frozen by liquid nitrogen can preserve MSCs,different generation of MSCs have a similar cell cycle.(2) Culture supernatant of glioma cells attract MSCs directional transfer.(3) Glioma cells can induce MSCs differentiate into endothelial-like cells by secret soluble cytokine,May be involved in the formation of tumor neovascularization,It may also be one of the mechanisms of MSCs targeted migrate toward gliomas.(4) MSCs express PDGF recptors,PDGF interact with its receptors, attract MSCs targeted transfer by concentration-dependent manner,May play a "chemokine-like" role in the process of MSCs selectively migrate to glioma.