Isolation and Identification of Rat Brain Microvessel Pericytes

【摘要】：Objective To explore the method of primary isolation,cultivation and identification of rat brain microvessel pericytes(RBMPC).Methods 10 brains were isolated from 3 week-old Wistar rats.Two-step enzyme digestions with one-step gradient centrifugation were used to isolate brain microvessel fragments.The microvessel fragments were seeded and cultured on 35-mm dishes.The RBMPC were identified according to the morphological observation under the Phase-Contrast Microscopy and the detection ofα-smooth muscle actin (α-SMA),NG2,vWF and glial fibrillary acidic protein(GFAP)associated antigens by Immunofluorescence method.MTT assay was applied to examine the growth curves of RBMPC.Results Migrating pericytes from cerebral microvascular fragments displayed by spreading with irregular projections and became confluent after 12-14 days.Pericyte markersα-SMA and NG2 associated antigens by Immunofluorescence method displayed positive,while vWF and GFAP by Immunofluorescence method were negative.The result proved that the cells were identified as RBMPC.The growth rate of primary cells was relatively slow.Passaged cells reached logarithmic growth phase after 36-60 hours and plateau phase after 72-108hours.Conclusion The above method could isolate high purity pericytes from rat brain microvessel successfully.We believe that exploring the properties of the primary culture of brain microvessel pericytes will be an important way to study cerebral microvascular function and the blood-brain barrier(BBB).