5B:CD133. dilution were all identified as CD133-positive by immunofluorescence. In serum-free medium, the proliferation of BTSCs in the ATRA group was observed significantly faster than that in the control group, but slower than that in the growth factor group and ATRA/growth factor group, and the size of the BTS in the ATRA group was smaller than that in the latter two groups(P< 0.01). In serum-containing medium, the expression percentages of CD133 and GFAP in the differentiated BTSCs were (2.29% 0.27%) and (75.60% 4.03%) respectively in the ATRA group, and (7.05% 0.49%) and (12.51% 0.77%) respectively Mouse monoclonal to CEA in the control group. The differentiation rate of BTSCs in the ATRA group was significantly higher than that in the control group (P< 0.05), but there was still CD133 expressed in the ATRA group. The differentiated BTSCs could re-form BTSs in serum-free medium. The percentage of BTS formation in the ATRA group was(4.84% 0.32%), significantly lower than that in the control group (17.71% 0.78%) (P< 0.05), and the time required for BTS formation in the ATRA group was (10.07 1.03)d, significantly longer than that in the control group (4.08 0.35)d (P< 0.05). == Conclusion == ATRAcan promote the proliferation and induce the differentiation of BTSCs, but the differentiation is incomplete, terminal differentiation cannot be achieved and BTSs can be formed again. == Introduction == All-trans retinoic acid (ATRA) is one of the strongest and most thoroughly studied differentiation inducers. It can induce the differentiation and apoptosis of a variety of tumor cells including glioma cells[1]. The concept of tumor stem cells Domatinostat tosylate suggests that the tumor stem cells are a cause of the formation, development and post-treatment relapse of tumors, as brain tumor stem cells (BTSCs) have a high potential of self-renewal and proliferation, which enables them to be resistant to chemo- and radiotherapies, so BTSCs must Domatinostat tosylate be eradicated in order to radically cure brain tumors. In this experiment, BTSCs are taken as the therapeutic target to study the effect of ATRA on the proliferation and differentiation of BTSCs, evaluating the antitumor activity of ATRA from a brand-new perspective. == Materials and methods == == 1 Major reagents and instruments == (1) Major reagents: DMEM/F12 and B27 were purchased from Gibco(U.S.A). Epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) were purchased from PeproTech (U.S.A.). ATRA,3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT), fetal bovine serum (FBS), trypsin, Cy3-labeled sheep anti-rabbit IgG and diamidino-phenyl-indole (DAPI) were all purchased from Sigma (U.S.A). Rabbit anti-human CD133 antibody was purchased from Abcam (U.S.A). Rabbit anti-glial fibrillary acidic protein (GFAP) antibody and FITC-labeled goat anti-rabbit IgG were purchased from Boster (Wuhan, China). (2) Major instruments: BB16 CO2incubator and HF-safe-1200 purifying worktable (Heraeus and Lishen company, Germany). CKX41 inverted phase contrast microscope, BX51 fluorescence microscope and imaging system (Olympus, Japan). ELISA Reader 2010 (Anthos, Austria). == 2 Experimental methods == (1) Isolation, culture and purification of BTSCs: The tissue samples were obtained from 3 surgical patients in Department of Neurosurgery, Anhui Provincial Hospital Affiliated to Anhui Medical University who had been diagnosed with glioblastoma during February-May, 2009. Fresh glioblastoma tissues without cystic degeneration, Domatinostat tosylate necrosis, calcification and electric coagulation were resected from the margin of tumor. By method in Ref[2], fresh glioblastoma tissues without cystic degeneration, necrosis, calcification and electric coagulation were resected from the margin of tumor, put in simplified serum-free medium (DMEM/F12, containing 2% B27, 20 g/L EGF and 20 g/L bFGF), and trimmed off necrotic tissues and residual blood vessels. After rinsing with serum-free medium for three times, the tissue masses were cut into pieces, disaggregated into single cell suspensions by using a Pasteur pipette (blowing repeatedly), and filtered with a sieve with the aperture of 74 m. The filtered single cell suspensions were stained with Trypan Blue. The living cells were counted, and primary culture was completed within 2 h, followed by inoculation in simplified serum-free medium (DMEM/F12, containing 2% B27, 20 g/L EGF and 20 g/L bFGF), and then culture.