Atg7 Regulates The Expression Of Fibronectin And Its Role In The Formation Of Blood-brain Barrier

Background and PurposeThe blood-brain barrier(BBB)is a structure composed of BMEC,basal membrane,pericyte and astrocyte(podocyte)with tightly connected structure,and the basement membrane plays an indispensable role in the assembly of blood brain barrier.The basement membrane is mainly composed of fibronectin(FN),lamina,collagen type IV,internal actin,and some glycoproteins.It is generally believed that FN can mediate adhesion between the base membrane and the surrounding cells.It plays an important role in the maintenance of the barrier of BBB,but the source of FN in the BBB membrane and the mechanism of its expression and regulation in the BBB membrane.It is not clear how to maintain the detailed mechanism in BBB.In recent years,more and more studies have shown that autophagy participates in the process of microvascular lesions in stroke,and the results of previous studies in my laboratory show that the key factor of autophagy-autophagy related protein 7(Autophagy-related protein 7,Atg7)plays an important role in the development of cerebral microvascular,but Atg7 and The relationship between the composition of BBB basement membrane has not been reported yet.The main purpose of this paper is to explore the relationship between the expression of the main components of the Atg7 and the BBB basement membrane by means of the model cells(human brain microvascular endothelial cells,HBMEC)and the conditional knockout of Atg7 vascular endothelial cells on the basis of observing the changes in the expression of FN during the development of cerebral microvessels,and to accumulate scientific data for further exploration of the homeostasis accumulation of BBB.Experimental Method 1.The expression changes of FN during the development of cerebral microvascular.1.1 Western blot was used to detect the expression of FN during brain development.2.The expression of FN and permeability of blood-brain barrier in mouse brain microvascular endothelial cells were detected by Atg7 conditional knockout mice.2.1 Preparation and identification of Atg7 conditional knockout mice.2.2 Brain concussion slices were made in Atg7 conditional knockout mice and wild control mice.2.3 The expression level of FN in endothelial cells was detected by tissue immunofluorescence.2.4 Mice were intraperitoneally injected with Evans blue,and the blood brain barrier permeability of Atg7 conditional knockout mice and wild control mice was detected by tissue immunofluorescence.3.Study on the relationship between Atg7 and FN expression by pattern cell(HBMEC).3.1 Construction of Atg7 stable HBMEC(human brain microvascular endothelial cell)cell line and control cell strain.3.2 The expressions of Atg7 and FN were detected by Real-time PCR,Western blot and cellular immunofluorescence staining.4.Preliminary study on the regulation of FN expression by Atg7.4.1 HBMEC was treated with signal pathway inhibitor,and the expression level of FN after different inhibitors was detected by RT-q PCR in order to determine the signal pathway involved in Atg7 regulation of FN expression.Results 1.The expression of FN in the brain of mice decreases with time.1.1 Western blot was used to detect the expression of FN in the brain of mice at 1,3,7,14,30 and 60 days after birth.The results showed that the expression of FN in the brain of mice decreased gradually with the increase of days after birth.2.In Atg7 conditional knockout mice,the expression of FN in brain microvascular endothelial cells decreased,and the permeability of blood-brain barrier increased.2.1 The expression of Atg7 in vascular endothelial cells was detected by tissue immunofluorescence staining with four month old endothelial cells knockout Atg7 mice and the same nest wild control mice.The results showed that compared with the wild control mice,CD31 localized the endothelial cells,and the FN fluorescence intensity of Atg7 conditional knockout mice decreased significantly.3.Interference with Atg7 results in decreased expression of FN in human brain microvascular endothelial cells.3.1 Real-time PCR was used to detect the mRNA expression level of Atg7 stable cells and control cells.The results showed that the expression of mRNA in FN was significantly lower than that of the control cell lines,after the interference of Atg7 expression.3.2 Western blot was used to detect the total protein expression level of Atg7 interfering cell lines and control cells.The results showed that compared with the control cell lines,the expression level of FN was significantly decreased after the interference of Atg7 expression.3.3 The cell immunofluorescence method was used to detect the expression changes in the extracellular matrix of the Atg7 stable interference cell line and the control cell group.The results showed that,compared with the control cell lines,the FN protein secreted to the extracellular matrix by Atg7 stable cell lines decreased significantly.4.NF-?B signaling pathway may be involved in the downregulation of FN expression induced by Atg7.4.1 The FN transcriptional level in human brain microvascular endothelial cells was detected by RT-qPCR under the signal pathway inhibitor.The results showed that the NFkappa B signaling pathway may be involved in the downregulation of FN expression caused by Atg7.ConclusionsCompared with the wild type control mice,the expression of FN in the endothelial cells of the endothelial cells with the conditional knockout of Atg7 decreased significantly,and the permeability of the blood brain barrier increased significantly.In vitro culture of HBMECs,interfering with Atg7 expression resulted in a marked decrease in mRNA and protein levels of FN,suggesting that Atg7 regulates FN expression at transcriptional level.NF-?B signaling pathway may be involved in the downregulation of FN expression induced by Atg7.