The Inhibitory Effects Of Celastrol On The Process Of Brain Glioma Stem Cells' Differentiation Into Vascular Endothelial Cell-like Cells

At present, the brain glioma stem cells theory has been more and more accepted by many scholars. Brain glioma stem cells (BGSCs) are seed cells of glioma,having stem cells'attributes such as self-renew, proliferation and multiple differentiation potential. In recent years, the researches have focused on glioma stem cells'differentiation into their parental phenotype cells called glioma cells both at home and abroad , but the studies on whether BGSCs cells can differentiate into vascular endothelial cell-like cells and then participate in tumor blood supply were less, so we studied on BGSCs'differentiation into vascular endothelial cell-like cells on certain conditions in vitro and application of celastrol inhibiting its differentiation process.The study involved two aspects of the glioma stem cells research:①brain glioma stem cells'differentiation into vascular endothelial cell-like cells on certain conditions in vitro.②the inhibitory effects of different concentrations celastrol on the differentiation process.PartⅠ:Brain glioma stem cells'differentiation into vascular endothelial cell-like cellsObjective To explore the feasibility of brain glioma stem cells'differention into vascular endothelial cells on certain conditions in vitro, and establish foundation for the further study of celastrol's inhibitory effect of the differentiation process.Methods Brain glioma stem cells were cultured in neural stem cell culture solution , then cells'growth changes were observed and CD133, Nestin ,CD31 and KDR immunofluorescence stainings were used to examine the cells after passage. Brain glioma stem cells were divided into the experimental group and the control group after passage. Cells of the experimental group were cultured by differentiation culture solution which included basal culture solution DMEM/F12 100ml, 10%FCS, VEGF 20ng/ml, EGF 20ng/ml , bFGF 20ng/ml, 1/100 N₂ in anaerobic environment which included 0%O₂,5%CO₂,10%H2 and 85%N₂, while the cells of the control group were cultured by ordinary culture solution which included basal culture solution DMEM/F12 100ml, 10%FCS in aerobic environment. Cells'growth changes were observed by the microscope. After 12 days, CD31 and KDR immunofluorescence stainings were used to examine the cells, and the cells were planted on Matrigel glue to observe the cells'growth changes.Result Brain glioma stem cells suspended in neural stem cell culture solution and the results of CD133 and Nestin immunofluorescence stainings were positive while the results of CD31 and KDR immunofluorescence stainings were negative. The experimental group cells were cultured by differentiation culture solution in anaerobic environment for 12 days. Flag stone phenomenon was observed. Then the results of CD31 and KDR immunofluorescence stainings were positive and tubular structures were observed after the cells were planted on Matrigel glue for 24 hours. While the flag stone phenomenon was not observed in the control group cells cultured by ordinary culture solution in aerobic environment for 12 days.And the results of CD31 and KDR immunofluorescence stainings were negative and tubular structures were not observed after the cells were planted on Matrigel glue for 24 hours.Conclusion The brain glioma stem cells which were cultured by differentiation culture solution in anaerobic environment in vitro for 12 days could differentiate into vascular endothelial cell-like cells.PartⅡ:The inhibitory effects of celastrol on the process of the brain glioma stem cells'differentiation into vascular endothelial cell-like cellsObjective To study the inhibitory effects of different concentration celastrols on brain glioma stem cells and the effects and influences on the process of the brain glioma stem cells'differentiation into vascular endothelial cell-like cells.Methods One part of brain glioma stem cells differentiated for 72 hours. And then celastrols of different concentrations such as 0.04μg/ml,0.2μg/ml,1.0μg/ml,5.0μg/ml,25.0μg/ml,125.0μg/ml were added to culture the cells for 48 hours. Cells continued to differentiate for 7 days.Then cells'growth changes were observed and CD31, KDR immunofluorescence stainings were used. The other part of brain glioma stem cells were planted on the Matrigel glue, and celastrols of different concentrations were added like the front steps. We observed whether tubular structures could form. In addition, the effects of celastrol on the proliferation of glioma stem cells were examined by MTT assay after celastrols were added for respectively 24 and 48 hours in the process of differentiation. Cells were collected after the different concentration celastrols took effects for 48 hours; Cell apoptosis was examined by AnnexinⅤ-PI assay; Cell cycle changes were examined by the cell cycle kit; And caspase-3 expression was examined by Semi-quantitative RT - PCR method.Result Only in 0.04μg/ml group, the flag stone phenomenon appeared, CD31, KDR immunofluorescence stainings were positive,and tubular structures could form on the Matrigel glue.Few tubular structures could be observed in 0.2μg/ml group cells.While the results were negative in other group cells. With the increase of celastrol concentration, the inhibitory effects on cells'growth were more stronger by MTT assay, cell apoptosis was more obvious by AnnexinⅤ- PI method;And caspase-3 expression was more obvious by semi-quantitative RT - PCR method.Conclusion The celastrol could inhibit the growth of the brain glioma stem cells and the process of the brain glioma stem cells'differentiation into vascular endothelial cell-like cells significantly in certain concentration range .The inhibitory process was in time-dose dependence. The mechanisms why celastrol could inhibit the process of brain glioma stem cells'differentiation into vascular endothelial cells probably may relate with the facts that celastrol could introduce cells apoptosis and block cells in S period.