The Role Of Gap Junction Channel On Vascular Outer Wall In Cerebral Vasospasm In Vitro

Objective:This experiment is in order to study the gap junction channel in vascular outer wall in vitro based on the earlier research, and simulate the structure of blood vessel outer wall by co-culture adventitial fibroblasts(AFB) with smooth muscle cells(SMC) on Polyethylene terephalate (PET). Simulates the in vivo subarachnoid hemorrhage's environment, Add the oxygenated hemoglobin which can not penetrate the PET membrane into the AFB,to observate the change of SMC's shape and Cx43 mRNA expression that the SMC has not contacted the Oxyhb. And then silencing the AFB's Cx43 gene by chemically synthesized siRNA transfection to observate the change of SMC's shape and Cx43 mRNA expression,the goal of the studies is to provide a basic evidence for the relation between the vascular outer wall gap junction information passageway and cerebral vasospasm.Methods :(1) Cell culture and identification of aorta thoracica's adventitial fibroblasts and basilar artery's smooth muscle cells. The media of rabbits' basilar arteries and aorta thoracica's adventitia were taken and cultivated with the explant attachment method. The cultured cells were identified with light phase contrast microscopy and immunocytochemical staining.(2) Smooth muscle cells(SMC) and adventitial fibroblasts(AFB) were grown on opposite sides of microporous polycarbonate filters(PET), to establish a SMC-PET-AFB co-culture cell model imitating the outer layer of vascular wall.(3) Co-culture AFB with SMC,then add the OxyHb into AFB's culture fluid. Detect the OxyHb's optical density in the culture fluid of AFB and SMC.(4) Scrape loading dye transfer (SLDT) and electron microscopy were used to detect cell gap junctional intercellular communication(GJIC) function...