Effects Of IgA1 Isolated From Henoch-sch?nlein Purpura Serum On The Binding Of Human Microvascular Endothelial Cells,its Proliferation And Migration Under Extracellular Acidification

ObjectiveTo study of serum IgA1 from Henoch-Sch,nlein purpura?HSP IgA1?on the binding of Human microvascular endothelial cells?HDMECs?to HSP IgA1,proliferation and migration of HDMECs and inflammatory cytokines stimulation under acidosis circumstance,to further clarify the mechanism of HSP vascular endothelial injury.Methods1.Collection of serum and Iga1 extraction from normal and HSP children10 children with acute HSP from November 2016 to February 2017 in our hospital were selected at the same age.Ten healthy children of the same sex were taken as the normal control group.The children in the control group were collected 5 ml of peripheral venous blood on an empty stomach every day after physical examination.The serum was separated by Jacalin affinity chromatography.2.The culture and experimental grouping of HDMECsThe cell culture bottle containing 15%fetal bovine serum?FCS?and RPMI1640medium was selected and incubated in 5%CO₂ incubator,36.5?,RPMI1640,and 3-5generation cells for experient.Pretest:?1?blank control group:15%fetal calf serum and RPMI1640 complete cell culture medium were cultured.Secondly,the normal IgA1 group was added to normal children's IgA1?250ng/L?and HSP group:the normal children's IgA1?250ng/L?was added,and 6h,12h and 24h were cultured respectively.According to the results of pre experimental groups as follows:blank control group:the culture medium for 12 hours in serum-free HDMECs;normal group IgA1:HDMECs in serum of healthy children with isolated IgA1?final concentration 250ng/L?in the culture medium 12h and HSP;group IgA1:HDMECs in HSP patients with isolated serum IgA1?final concentration 250ng/L?in the culture medium 12h;pH6.5 group:6h culture medium HDMECs in 15%fetal bovine serum,regulating the extracellular fluid pH value to 6.5 6h;group IgA1+pH6.5:HDMECs in the HSP containing HSP in children with IgA1?final concentration 250ng/L?training6h medium,the regulation of extracellular fluid pH value to 6.5 6h.3.The amount of combination of IgA1 and HDMECsThe cells were divided into blank control group,normal IgA1 group,HSP IgA1 group,HSPIgA1+pH6.5 group and HSPIgA1+pH6.5 group.The combination of IgA1 and HDMECs under different conditions was collected by flow cytometry.4.Apoptosis and migrationMTT were used to acess the proliferation of HDMCEs,cell scratch test and Transwell compartment were used to detect cell migration distance and number.5.Inflammatory factor determinationThe cells were collected after 12h culture,and the supernatant of the cells was collected.The concentration of IL-8 and ET-1 in the supernatant of the cells was detected by enzyme linked immunosorbent assay?ELISA?.Result1.HSPIgA1 can lead to cell apoptosis,with the increase of time,when the 12h number is the largest explosion of apoptosis2.Serum IgA1 could bind to endothelial cells,and extracellular acidification could promote the binding of IgA1 to HDMECs.Compared with HSPgA1 alone,the binding positive cells were significantly increased?P<0.01?.3.Compared with normal IgA1 group,serum IgA1 could inhibit HDMECs proliferation?P<0.05?and promote migration?P<0.05?.Compared with HSPIgA1group,HSPIgA1 PH6.5 group further inhibited cell proliferation?P<0.05?,cell migration distance and migration cell number increased significantly?P<0.05?.4.Compared with normal IgA1,serum IgA1 could increase the level of interleukin-8?IL-8?ET-1 secreted by endothelial cells,which was obviously increased under extracellular acidification,that is to say,the level of IL-8+ET-1 secreted by HSP IgA1 alone was significantly higher than that of HSP IgA1 alone?P<0.01?.Conclusions1.Serum IgA1 could bind to vascular endothelial cells,induce apoptosis of endothelial cells,increase the concentration of migration inflammatory factors,and induce cell damage.Extracellular acid condition further promoted the deposition of vascular endothelial cells by IgA1.The inflammation reaction of vascular endothelial cells is aggravated and the injury of vascular endothelial cells is aggravated.2.The mechanism of vascular endothelial injury in children with HSP may be associated with gA1 through binding with vascular endothelial cells to induce immune inflammatory response,promote the increase of vascular endothelial cell permeability,lead to vascular endothelial cell injury,and then produce acidic microenvironment in vivo.This acidification may further aggravate vascular endothelial damage in children with HSP.