Effects Of Glioma Cells On Blood-brain Barrier And Their Molecular Mechanisms

Objective: A well-controlled local enviroment is very important for the function of brain. The mechanism that controls this stable internal enviroment within the brain is known as blood-brain barrier. As the most common neoplasm in central nerve system, glioma cells have a great impact on BBB and can disturb the normal function of BBB, which lead to many neurological symptoms and complications associated with glioma. However, up to date, we know little about how glioma cells affect BBB and what are the underlying molecular mechanisms. In this research, we used an in vitro BBB model to investigate the impacts of glioma cells on the blood-brain barrier and their molecular mechanisms.Methods: l.A blood-brain barrier model was established by coculture of ECV304 and astrocytes in vitro; 2. The activity of Y -glutamyl .transpeptidase( y-GT) and alkaline phosphatase(ALP) were measured by Beckman Clinical Systems; S.Morphological change of tight junctions in the blood-brain barrier was determined by silver staining; 4.The function of tight junctions of in vitro BBB was studied by Millipore-ERS system; 5. The expression levels of zonula occluden l(ZO-l), caveolin-1 and caveolin-2 were analyzed by semiquatitative RT-PCR before and after dexamethasone treatment; 6. The expression level of occludin in endothelial cells was determined by Western Blotting; 7-HPLC andwere used to determine the change of water transport of in vitro blood-brain barrier model after the influence of glioma cells with or without dexamethasone; 8.The expression levels of aquaporin-1 and aquaporin-4 were analyzed by semiquatitative RT-PCR before and after dexamethasone treatment; 9-HPLC technique was used to determine the extracellular glutamate concentration to the abluminal side of blood-brain barrier; lO.The expression level of EAAT3 was analyzed by semiquatitative RT-PCR.Results: 1-The blood-brain barrier in vitro has a similar ultrastructure with BBB such as tight junctions between endothelial cells, lots of mitochondria and little pinocytosis. The in vitro BBB had a high TER value of 321.3 Q cm2 after coculture for lOd. The activity of Y - GT and ALP of ECV304 were increased and could reach 480.24+104.17 U/mg protein, 589.13 + 54.35 U/mg protein after coculture with astrocytes; 2.The TER of BBB was decreased to 213.7 +56.2 cm2 after coculture with glioma cells for 7 days; After dexamethaxone treatment, the decreased TER was improved and increased to 275.3 +48.1 cm2; S.Lots of tight junctions in BBB were showed to be disrupted by glioma cells; however, some intact tight junctions could still be found in some area; After dexamethasone treatment, the quantity of intact tight junctions was increased; 4.The expression levels of ZO-1, caveolin-1 and caveolin-2 in endothelial cells were decreased after the impacts of glioma cells, which could be improved by dexamethasone treatment; 5.Western blotting analysis showed that occludin expression level in endothelial cells was decreased by glioma cells and glucocorticoid could improve the decreased expression level of occludin; 6. Glioma cells could induce the abnormal aquaporin-1expression in endothelial cells and decrease aquaporin-4 expression level in astrocytes. At the same time, the water transport of in vitro blood-brain barrier model from luminal side to abluminal side was increased after coculture with glioma cells. Compared with those results, dexamethasone could decrease the abnormal aquaporin-1 expression in endothelial cells and increase the decreased aquaporin-4 expression level in astrocytes caused by glioma cells. As the result, the water transport from luminal side to abluminal side was decreased; 7.The extracellular glutamate concentration to the abluminal side of normal blood-brain barrier was 14.487+ 1.755 u mol/L; The extracellular glutamate concentration after the influence of glioma cells increased to 22.609+2.124 u mol/L; After dexamethasone treatment, the extracellular glutamate concentration was 19.906+1.934u mol/L. At the same time, the EAAT3 expression level of en...