Culture Of SD Rat Cerebral Artery Endothelial Cell

BackgroundAngiogenesis is the process of formation of offspring blood vessels,through endothelial cells proliferation or endothelial cells migration to somewhere.Cerebral artery can reconstruct blood supply effectively and prevent from the occurrence of dementia and cerebral infarction,which were caused by multiple and diffuse cerebral atherosclerosis.The study on Gene function and signal transduction mechanism about embryonic cerebral vascular disease has laid a theoretical foundation for the study of cerebral angiogenesis in recent years[1].Yoshimura S inject the Carrier which contains complex gene of liposome-HVJ with hepatocyte growth factor into the subarachnoid space in rats.we discovery that blood vessels renews on the surface of the brain.and the cerebral perfusion volume increased significantly.Therefore we think that the hepatocyte growth factor could promot angiogenesis in the brain[2].In recent years,the research results about the mechanism of the artery have been made progress continuously[3,4,5,6].In Chinese and foreign language database were not found in vitro cultivation method of SD rat CAEC,which brought certain difficulty to the related experimental study.Although the methods of SD rat brain microvascular endothelial cells(BMECs)culture were mature,but the cerebral artery endothelial cell(CAEC)was different from brain microvascular endothelial cells(BMECs),the cerebral artery endothelial cells constitute the blood-brain barrier was not involved,and the function of the two cells showed different heterogeneity.The cultivation of SD rat cerebral artery endothelial cell for in vitro study mechanism of cerebral arteries new study provides the basis.The SD rat cerebral artery endothelial cells in vitro culture was difficult,we had passed through domestic and foreign cell culture methods for analysis and trial and error,and summed up the method that could cultivated high degree of purification of SD rat cerebral artery endothelial cell.ObjectiveTo explore the method of culturing highly purified primary SD(Sprague-Dawley)rat Cerebral Artery Endothelial Cell and identified in vitro.We determination the cerebral artery endothelial cells proliferation in rat.We check the ability of cells migration throughcells scratch test.MethodsThe brain cerebroarters of 5 to 6 weeks old male SD rats with 110 to 140 grams were selected,mincing into pieces.They were incubated into gelatin-coated 35 mm dish plates with ECM.We observed the shape and growth status of Endothelial Cells under the ordinary option microscope.Immunofluorescence assay the expression of artery endothelial markers Ⅷ factor related antigen.The effects of migration of CAECs were measured by the MTT assay,Scratch wounds experiment.ResultsAfter 60h culture,it was visible that the cells grew out from cultured cerebral artery pieces.Seven days late,the cells grew in confluence locally and the morphology exhibited "cobble" pattern.Immunofluorescence staining showed that most cells were factor Ⅷpositive.The purity of CAECs was 90%.1.After 60h culture,it was visible that the cells grew out from cultured cerebral artery pieces.Seven days late,the cells morphology exhibited "cobble" pattern “.Seven days late,they became 80% confluent and displayed uniformly.1:3 after passage 24 h visible Petri dish adherent spindle cell in bulk.After passage 6 D full passage can continue.6generations of cells proliferation and morphology did not change..2.Immunofluorescence and immunohistochemical staining showed that most cells wer e factor Ⅷ positive.PBS instead of adding anti set the negative control staining was negati ve.Immunofluorescence cell related antigen staining,the cytoplasm green nuclei were stai ned blue.The purity of CAECs was 90%.3.We determined the growth curve of CAECs by MTI assay.The cleavage speed of CAECs at day 4-8 after passage was faster,while at day 9-12 after passage.the cleavage became obviously slower.,which might be the result of contact inhibition..ConclusionThis method can successfully obtain the primary CAECs with high purity.