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The Research of Tat Peptide-Assisting USPIO Crossing the Blood Brain Barrier Model

Author: YinBo
Tutor: ZhangSuMing
School: Huazhong University of Science and Technology
Course: Neurology
Keywords: Primary culture Astrocyte Brain microvascular endothelial cell co-culure Blood brain barrier Blood-brain barrier model Tat peptide Superparamagnetic iron oxide particles Fluorescence labeled
CLC: R-332
Type: Master's thesis
Year: 2010
Downloads: 66
Quote: 0
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Abstract

Objective To structure blood brain barrier in vitro using brain microvascular endothelial cells and astrocytes.Methods 1.primary culture of astrocytes:cerebral cortex was abtained from Kunming white old-fetal mice,treated with trypsin,and filter.Then remove fibroblasts with differential attachment,and eliminate oligodentrocytes and microglial cells with orbital shaker.Check the cells with immunostaining for glial fibrillary acidic protein(GFAP);2.primary culture of brain microvascular endothelial cells:cerebral cortex was obtained from 1 to 3 days new-born Kunming white mice,digested by collagenaseⅡ,and treated with BSA gradient centrifugation and second enzyme digestion of collagenase/dispase.Then change the culture solution in the next 24h to pury the cells. Checked by immunostaining forⅧfactor associated antigen;3.co-culture passageⅡbrain microvascular endothelial cells and passageⅣastrocytes by transwell inserts.Evaluate the Brain Blood Barrier model by AKP transpeptidase activity,4h permeation test,and the permeation of Horseradish Peroxidase(HRP).Results passageⅣAstrocytes was characterized by GFAP immunostaining, and positive rate was 95%;passageⅡbrain brain microvascular endothelial cells was characterized byⅧfactor immunostaining,and positive rate was 90%.The AKP activity of microvascular endothelial cells was higher in co-culture model than monolayers.Conclusion We can establish stable blood brain barrier model in vitro,which lay the foundations for further research of opening the BBB.And it’s also a good model to tentatively identify the permeation of drugs for diagnosis and treatment. Objective To detect whether tat peptide-assisting superparamagnetic iron oxide particles (Tat-MNPs) can pass the blood-brain barrier.Methods Using transwell co-culture construct blood-brain barrier model in vitro.With a different dilution of FITC labeled Tat peptide-mediated superparamagnetic iron oxide particles (FITC-Tat-MNPs) joined the transwell for the pool, in the different time periods, using inverted fluorescence microscope by the pool if there are fluorescent display, to determine the FITC-Tat-MNPs can pass the blood-brain barrier model.Results different levels of green fluorescence were visible on PET membrane after the 4h FITC-Tat-MNPs joined in, and relatived with FITC-Tat-MNPs diluted multiples,and 4d after the green fluorescence can be seen in the cells at the bottom of six plates.Conclusion FITC-Tat-MNPs may be through the blood-brain barrier model, and maybe crossed by penetrating cells, it provided a good carrier of nuclear magnetic resonance technology for early diagnosis of Alzheimer disease.

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