Research Of Establishing In Vitro Blood- Brain Barrier And Antibiotics’ Permeability Of In Vitro Blood-brain Barrier

Objective:By screening a new antibiotic with strong antibacterial activity in vitro but undefined blood-brain barrier permeability (linezolid, biapenem, sulbactam, tigecycline) and traditional antibiotics treating intracranial infection (vancomycin, meropenem), through human umbilical vein endothelial cells (HUVEC) and mouse glioma cells (C6) blood-brain barrier in vitro co-culture construct (Blood-Brain Barrier, BBB) cell model for drug through experiments of the blood brain barrier in vitro, select the appropriate antibiotic or combination in order to provide a theoretical basis for clinical treatment of central nervous system infections.Methods:1, Using employer umbilical vein endothelial cells (HUVEC) and mouse glioma cells (C6) through non-contact co-culture in vitro blood-brain barrier constructed (Blood-Brain Barrier, BBB) cell model, evaluate the blood-brain barrier (BBB) function by measuring transendothelial impedance (TEER).2. Choose new antibiotics with strong antibacterial activity in vitro but the blood-brain barrier permeability is not yet clear (linezolid, biapenem, sulbactam, tigecycline) and traditional antibiotics treating intracranial infection (vancomycin, meropenem),after appropriate treatment, configure 0.05,0.1,0.2,0.4,0.6mg/ml series of the concentration of standard solution. Taking the above series of concentrations of the drug each 20ul, add blank broth and configure to 0.005,0.01,0.02,0.04,0.06mg/ml series of concentration of the drug, after pretreatment the culture medium, Using high performance liquid chromatography (HPLC),peak area (X) of the drug concentrations to do linear regression (Y), obtained the regression equations and correlation coefficients (R2). Take each one of the six drug lml and add them to the in vitro blood-brain barrier (BBB) cell donor pool model 0.05mg/ml respectively, after training 30 min,1,2,4 h, take part of the receptor pool culture respectively, when the culture pre-treatment was finished, use HPLC to determine the actual drug concentration in the culture medium, comparing the actual culture medium after 4h drug concentration with the initial drug concentration to measure the in vitro blood-brain barrier (BBB) permeability of the six antibiotics model.RESULTS:1, Through the measure of transendothelial impedance (TEER) of blood-brain barrier in vitro cell model, HUVEC and C6 can be successfully constructed in vitro co-culture of BBB (BBB) model was proved and they can meet the needs of the follow-up test.2, Six kinds of antibiotics show good linear in 0.005-0.6mg/ml range, and meet the test requirements. Vancomycin at 30min,1h,2h,4h actual culture medium at a concentration of 0.0015mg/ml,0.0026mg/ml,0.0039mg/ml,0.0048mg/ml,4h after in vitro BBB permeability of 9.6%. Linezolid at 30min, 1h,2h,4h actual culture medium at a concentration of 0.003mg/ml,0.0047mg/ml,0.0075mg/ml,0.0104mg/ml, 4h after the transmittance was 20.8%. Biapenem at 30min, 1h,2h,4h actual culture medium at a concentration of 0.0012mg/ml,0.0021mg/ml,0.0028mg/ml,0.0037mg /ml,4h after the transmittance of 7.4%. Meropenem at 30min, 1h,2h,4h actual culture medium at a concentration of 0.0014mg/ml,0.002mg/ml,0.0037mg/ml,0.0052mg/ ml,4h after the transmittance was 10.4%. Sulbactam at 30min, 1h,2h,4h actual culture medium at a concentration of 0.0015mg/ml,0.0027mg/ml,0.0044mg/ml,0.0051mg /ml,4h after the transmittance was 10.2%. Tigecycline at 30min, 1h,2h,4h actual culture medium at a concentration of 0.0043mg/ml,0.0047mg/ml,0.0056mg/ml, 0.0062mg/ml,4h after the transmittance was 12.4%.CONCLUSION:1, HUVEC and C6 co-culture can be successfully established in vitro cell model which can reflect the blood-brain barrier.2,Compared with vancomycin, linezolid has maximum permeability in vitro blood-brain barrier.Linezolid can be the best choice to treat intracranial infection of Gram-positive bacteria. Meropenem, tigecycline and sulbactam in vitro blood-brain barrier permeability is closed, overdose tigecycline can be an alternative treatment of multidrug-resistant Gram-negative bacilli intracranial infection, and Sulbactam joint meropenem therapy may be an effective choice of multiple intracranial resistant Acinetobacter baumannii infections.