Mechanism Of Calmodulin Regulating Endocytosis Of Cerebral Microvascular Endothelial Cells

Objective: The dysfunction of blood-brain barrier is one of the pathological signs of ischemic stroke.As an important component of blood-brain barrier,brain microvascular endothelial cells mediate the selective passage of substances inside and outside blood vessels through its endocytosis,thus maintaining the steady permeability of blood-brain barrier.Calmodulin is a calcium ion sensor widely expressed in eukaryotic cells,which is involved in the regulation of neuronal endocytosis.This study took human brain microvascular endothelial cells as the research object to explore the mechanism of calmodulin regulating the endocytosis of brain microvascular endothelial cells.Method: 1.Using si RNA to construct human brain microvascular endothelial cells with transient knockdown of calmodulin;2.The uptake of Alexa Fluor488 labeled transferrin by human brain microvascular endothelial cells was analyzed by live cell real-time imaging technology,and the difference of endocytosis between control cells and calmodulin knockdown cells was compared.3.RT-q PCR and Western blotting were used to detect the changes of m RNA and protein levels of Dyn family members in human brain microvascular endothelial cells after knocking down calmodulin;4.RT-q PCR,Western blot and immunofluorescence techniques were used to detect the changes of the level of clathrin m RNA and protein in human brain microvascular endothelial cells after knocking down calmodulin;5.The changes of the distribution of clathrin protein on the surface of human brain microvascular endothelial cells after knocking down calmodulin were detected by TIRF microimaging technique;6.The uptake of fluorescent-labeled glucose analogue 2-NBDG by human brain microvascular endothelial cells was analyzed by flow cytometry,and the effect of knocking down calmodulin on glucose uptake was compared.7.RT-q PCR and Western blotting were used to detect the changes of m RNA and protein levels of GLUT family members in human brain microvascular endothelial cells after knocking down calmodulin;Results: 1.The uptake of transferrin and endocytosis of human brain microvascular endothelial cells decreased after knocking down calmodulin;2.After knocking down calmodulin,the expression of Dyn2 m RNA and protein in human brain microvascular endothelial cells decreased;3.After knocking down calmodulin,the expression of clarin m RNA and protein in human brain microvascular endothelial cells decreased;4.After knocking down calmodulin,the distribution of clathrin on the surface of human brain microvascular endothelial cells decreased;5.The uptake of glucose analogues by human brain microvascular endothelial cells decreased after knocking down calmodulin;6.After knocking down calmodulin,the expression of GLUT1 m RNA and protein in human brain microvascular endothelial cells decreased.Conclusion: In human brain microvascular endothelial cells,knockdown of calmodulin leads to decreased expression of endocytosis process-related proteins Dyn2 and clathrin,thus weakening the endocytosis function of endothelial cells.2.In human brain microvascular endothelial cells,knockdown of calmodulin results in a decrease in glucose uptake mediated by transporter GLUT1.