| Objective: To establish an in vitro model of blood-brain barrier (BBB) and examine its biological functions. This model could be a useful tool for further study of related diseases in vitro.Methods:(1) Brain microvascular endothelial cells (BMECs) were isolated from neonatal Sprague-Dawley rats by two-step filtration, collagenase digestion and density gradient centrifugation using low-molecular weight dextran. After 4-hour seeding, the floating cells were washed away and the adherant BMECs were kept. The cell morphology was observed under phase-contrast microscope. The purity of BMECs was examined by immunohistochemistry staining. The contamination of microvessles was determined by the expression of AKP by BMECs. The viability of BMEC was determined by ET-1 releasing. (2) Type-1 astrocytes were obtained by shaking culture and were identified by the expression of glial fibrillary acidic protein (GFAP). (3) Type-1 astrocytes were seeded on the bottom of the Transwell polyester-multipore-membrane, which coated with rat tail collagen at a density of 2.5 X lOVcm2. The cells were allowed to attach for 4hrs, and then turned over the filter into normal position, cultured for 7 days.The confluent BMECs at passage 2 were plated on the inner side of the Transwell.The medium used for the coculture was replaced with DMEM/F12 medium without serum. Under these conditions, BMECs form a confluent monolayer in 7 days.BMECs were observed by HE staining.This model was further examined its biological functions.Enzyme activity of Y -glutamyl transpeptidase( Y -GT) was determined with a diagnostic assay kit (as U/mg of protein). (MW: 4kDa) The endocytosis of Fluorescen-dextran by BMEC was measured; Restrixtion of paracellular transport was determined by analysis of the apparent permeability coefficent (Papp) for sodium fluorescein (FLU MW:376KDa);125I-BSA(MW: 66kDa) that passed the polyester-multipore-membrane was then counted in scintillation counter at various time points.(4) The co-culturing of human umbilical vein cells(HUVEC) and T1A was also established and FLU ^ 125I-BSA was observed.Results: (1) The purity of cultured BMECs was 90% using our modified method. Polygonal or cobblestone like cells were observed under microscope, and cells are adherent, proliferated in a monolayer. The cultured BMECs were positive for VIIIF-Ag with immunohistochemistry staining. The purity of positive stained cells was 90%. The percentage of AKP-positive cells was 93%, with a total score of 112. The primary BMECs released 388.03pg/ml of ET-1 and the passage 2 cells secreted 591.75pg/ml. (2) After shaking culture, the purity of TIAwas 98%.T1Awas positive for GFAP with immunohistochemistry staining. (3) Co-cultured BMECs showed characteristic morphology of endothelial cells.The specific activity of Y -GT in primary BMECs was 16.42 U/mg cell protein, whereas in BMEC passage 2 it was 3.69 U/mg cell protein.The enzyme activity in BMEC and T1A coculture is 11.3 U/mg cell protein (n=6). The Y -GT levels increased after coculturing.The Papp of paracellar transport marker FLU across the BBB model in vitro is 9.0 + 1.022 X 10-6cm/s.Co-culturing of astrocytes did not affect the rate of pinocytosis.Our BBB model showed saturation to BSA which transported BBB by receptor.(4) Pappof FLU across the BBB model by HUVEC and T1A is 15.22 + 3.42 X 10-6 cm/s.The co-culturing of HUVEV and T1A also provided a barrier to the diffusion of [125I]-BSA, but it did not show saturation to BSA.Conclusion: (1) The Rat BMECs were successfully cultured in vitro by two-step filtration, collagenase digestion and density gradient centrifugation in our study. Our method of isolation and culture of BMECs was easier and more efficient than others. This protocol was established for an in vitro model of BBB. (2) This system we established sustained the morphological and enzymatic characteristics of BBB in vivo. This model restricted paracellar and receptor-dependent transport marker through BBB. However, there was some difference between in vitro and invivo...
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