Establishment Of A Stable Cell Line Of Glioma Stem/progenitor Cells With RFP Infection By Lentivirus And Observations Of Their Biological Properties

0bjective To understand the feasibility and methods of red fluorescent protein(RFP)transfection in glioma stem cells by RFP-Lentivirus vector HIV-1．Methods The gliomastem cells were cultivated in serum free medium,then,immunostained with stem cellsurface marker antibodies-CD133, Nestin. The human brain glioma stem cells wastransfected with the lentivirus vector HIV-1containing red fluorescent protein(RFP)gene．Cells expression of RFP were observed by fluorescent microscopy；Flow cytometricsorting red fluorescent expression of gliomas stem cells， the sorting of the human brainglioma cloned stem cells under the continuous culture for20generations, the flowcytometric detected transfection efficiency of RFP cells;Cell proliferation of RFPuntransfected and transfected group were assayed by MTT method; Through the PCRamplification detection red fluorescent (RFP) gene integration to stem cell genome; Andthen analysed following biology characteristics of RFP cells: Cells immunohistochemicaldetected stem cell surface markers CD133, Nestin expression; Cancer stem cells havemultiplex differentiation potential, so induced it to differentiation of glial and neurons cellsin blood serum medium for10days, and Cell immunohistochemical was used to detect theexpression of GFAP and S100,which were glial cells markers; we cultivated RFP cells inendothelial differentiation medium for10days and to observe the changes of appearance.We also cultivated these cells on Matrigel for10days, and the process of morphologicalchanges was observed. Ten days later, part of the Matrigel containing cells was detectedfor the expression of CD31by immunohistochemistry; we Collected stem cell with redfluorescent protein expression transplantation to green fluorescent nude mice subcutaneous,tumors were stained with hematoxylin and eosin for histopathological examination. Thenwe could observe growth way, red fluorescent expression, cells form and tumor bloodessels of RFP cells in GFP nude mice.Results After glioma stem cells were transfected byRFP-Lentivirus vector HIV-1and continuous passage cultured in vitro．RFP gene stablyhigh express in glioma stem cells.Compared with RFP untransfected group，the cell growth curve of RFP transfected group had no significant difference(P>0．05), RFPtransfected group still express stem cell surface marker. RFP expression did not interferewith the biological properties of the transformed cells compared to the parental cell line.We also demonstrated that lentiviral-transduced glioma stem cells maintained stablehigh-level RFP expression during their growth in vitro and in vivo.Conclusion RFP can behighly effectively transfered into glioma stem cells under HIV-1lentivirus vectormediation, and have no influence to cell growth．...