Empirical Study Of Differential Expression Protein On Cerebral Vasospasm In Rabbits

Objective:Cerebral Vasospasm(CVS) is the most serious and common complication after subarachnoid hemorrhage(SAH).The mechanism is still unknown and without special treatment therapies.This study used proteomics research methods,two-dimensional echocardiography(2-DE)and MALDI- TOF/TOF,to identify differentially expressed proteins of SAH and use western blotting validate differential expression protein.To investigate molecule mechanism of CVS and search clinical application potential molecule target point settle groundwork. Expect through this study can identify CVS mechanism and search significant cure methods.Motheds:1. Establishing rabbit's CVS model after secondary SAH:Twenty four New Zealand White rabbits random fractionated normal control group (C) and CVS group (T), each group fractionated three portion:C1,C2,C3 and T1,T2,T3 each portion have four rabbits. Use rabbit twice SAH model.The control group also punctured but did not injected arterial blood.2. Brain angiography and morphological observation:Doing DSA after and before the model established and comparing basilar artery diameter's changes in different phases. After the second angiography in 3d, the heart of total cerebral perfusion is fixed as HE staining and observes the morphological changes under light microscope.3. Two-dimensional electrophoresis, silver staining and mass spectrometry identify:Animals in each group for extracting brain, separating brain arteries and extracting protein after DSA.2-DE and mass spectrography analysis technique detection and identification BA protein express changes during CVS.4. Western Blotting validate differential protein. Select arginase which is high expressed in CVS as target protein. use western blotting to detected Cx43 express changes after added arginase blockade.Experiment fractionated normal control group,SAH-3d group,Arginase+SAH-3d group. Results:1. Successfully established rabbit's CVS model after secondary SAH:DSA demonstrates that experiment group 3d compared to Od showed basilar artery contraction significant (64.2%±0.5% P<0.01)。control group 3d compared to Od showed basilar artery diameter without significant difference (89.3%±8.1%).Light microscope showed control group cerebral vessels endothelial cell homogeneous distribution, nuclear ellipse, light dye; its inferior inner elastic layer smooth; smooth muscle cell distribute uniformity,more compact; SAH group cerebral vessels endothelial cell nuclear chromatin assemble; interior elasticity membrane show ripple appearance significant; smooth muscle cell distribute rarefaction, surrounding red dye tissue mult significant, nuclear long fusiform shape, between interior elasticity membrane and smooth muscle layer have dropsy.2. Two dimensional gel electrophoregram analysis:Use imagemaster5.0 software to analyze CVS and normal basilar artery tissue two dimensional gel electrophoregram, choose three glue repeat appear spot of same group participate compare, By means of volume percentage as comparison value, choose gray scale volume(I.e. express content) relative value above 1.5 times spot as difference spot.Gain differential expression protein spot number together is forty-nine.Among the total fourteen spot high expression at normal basilar artery,thirty-five spot high expression at CVS.3. Mass spectra identification and data base querying result:Use mascot search in SWISS-PROT database to identify all forty-nine difference protein spot, thirty-five differential expression protein spot was identified among the total:Twenty-four high express at CVS and eleven low express at CVS.4. Arginase blockade can significant prevented the enhancement of Cx43 after CVS.Conclution:Established 2-DE patterns of rabbit spasm basilar artery and paired normal basilar artery and 35 differentially expressed proteins spots were identified. Which may participate cerebral vasospasm. Arginase may participate CVS through gap junction.