Analysis Of Chemoresistance Of Brain Tumor Stem Cells In U251 Sorting By MACS

OBJECTIVE The aim of the first part was to sort subpopulation of CD133+ and CD133- cells in the human Glioblastoma Multiforme(GBM)U251 cell line.To explore the difference of proliferation,differentiation and tumorigenic potential in vivo between the two subpopulation.The cytobiologic character of CD133+ subpopulation was the emphasis of this study;To investigate the specificity of CD133 to be specific surface marker(SSM)of brain tumor stem cells(BTSC)and the specificity and efficiency of the system of FACS sorting BTSC.The purpose of the second part was to compare the difference of drug sensitivity between the two subpolulation,and analysis the mechanism of chemoresistance by comparing the different expression of gene of ABC transporter,DNA repair and apoptosis.METHODS In the first part,CD133+ and CD133- subpopulation cells in the human GBM U251 cell line were sorted by MACS.Single isolated CD133+ and CD133- cells was cultured in serum-free DMEM/F12 medium,containing B27(1∶50),basic fibroblast growth factor(bFGF,20μg/L),epidermal growth factor(EGF,20μg /L)or medium containing 10%fetal bovine serum.Cell cycle of the two subpopulation was analyzed by flow cytometry(FCM).MTT and monoclone forming rate(MFR)assay was performed to investigate the capacity of self-rewal and clonogenic potential of the subpopulation cells. Immunofluorescence staining was performed to investigate the multidiferentiation of CD133+ cells and brain tumor sphere(BTS), formed by CD133+ cells cultured in serum-free medium.At last,in order to investigate the tumorigenic potential of the two subpopulation in vivo, xenograft in BALB/c nude mice brain was performed.On the foundation of cell-sorting,the second part was aimed to analyze drug sensitivity of the two subpopulation,the cell morphology change was examined by phase microscopy after treated with anti-tumor drug.MTT and flow cytometry was performed to analyze the drug sensitivity of the two subpopulation to anti-tumor drug,Teniposide,Carmustine and Cisplatin. Finaly,ABC transporter,DNA repair and apoptosis gene expression of the two subpopulation was analyzed by RT-PCR.RESULTS The first part show that,only4.5%cells was CD133+ in the total popul- ation of U251 cell line,and the purity of CD133+ cells in the CD133+ population can reach 92.4%after cell-sorting.The single CD133+ and CD133- cell was cultured in serum-free DMEM/F12 medium,containing B27(1∶50),basic fibroblast growth factor(bFGF, 20μg/L),epidermal growth factor(EGF,20μg /L)and serum-contain medium respectively.The CD 133+ cells exhibited powerful proliferation capacity.The single CD133+ cells could proliferate and form typical brain tumor sphere(BTS),while CD133- cells couldn't.And the cell growth curve showed that CD133+ cells proliferated remarkably faster than CD133- cells while cultured in serum-free medium.The cell proliferation index of CD133+ and CD133- cells was 40.1%and 30.6% respectively,showed by FCM.And the MFR of CD133+ and CD133-cells was 78.5-92.4%and 0.8-2.4%respectively.Immunofluorescence staining showed that,CD133+ cells and the BTS,forming by single CD133+ cells,could be induced to differentiate into mature neurocyte cells,which express NeuN and GFAP in the serum-containing medium. Xenograft assay showed that,CD133+ cells had tumorigenic potential in vivo,while CD133- hadn't.In the second part of this study,apoptotic morphologic change of CD133- cells was obvious in present of anti-tumor drug,while CD133+ cells could maintain survive.Anti-tumor drug assay showed that,CD133- cells were more sensitivite to Teniposide, Carmustine and Cisplatin than CD133+ cells.Treated with Teniposide, apoptotic percentage of CD133- cells were remarkerly higher than CD133+ cells,and "sub-Glpeak" appeared in CD133- subpopulation while CD133+ did not,showed by FCM.And FCM also showed that, accumulation of anti-tumor drug in CD133+ cells remarkably lower than CD133- cells.RT-PCR showed that,CD133+ cells expressed remarkably higher than CD133- cells in MDR1,BCRP,MRP1,MGMT and Bcl-2.CONCLUSION The U251 cell line contained a small proportion of CD133+cells,which had the capacity of proliferation,multidifferentiation and tumorigenic potential in vivo.The subpopulation of CD133+ cells was the brain tumor stem cells subpopulation in U251. MACS was an optimizing cell sorting system in BTSC sorting.The subpopulation of CD133+ cells was the chemoresistant subpopulation in U251 cell line.High expression of ABC transporter,DNA repair and apoptosis maybe the important chemoresistance mechanism of CD133+ cells.