| BackgroundsCryptococcus neoformans is an opportunistic pathogens,generally distributed in the environment in the soil,not alien to the human body?In recent years,cryptococcosis cryptococcal new violations of human patients each year worldwide total of up to one million people,especially after the violation of the human brain blood-brain barrier,a high mortality rate,treatment is more difficult.Human brain microvascular endothelial cells in a class of protein,S100A10 protein is a calcium-binding protein family in one of S100 proteins,molecular size of 11 KD,also known as p11 protein.In the cell nucleus and have the presence of liquid,often as a annexin A2 binding transmembrane proteins play an important role in maintaining normal cell physiological activities.The human body environment the blood system is not activated plasminogen contain traces,and endothelial cells S100A10 protein can bind not activated plasminogen protein,plasminogen protein by altering the steric like,play catalytic it's easier to urokinase(UPA)to activate the role of the bridge.The study also showed that incubation of cryptococcal and brain microvascular endothelial cells,S100A10 in mRNA levels increased.S100A10 spatial location and position of urokinase receptor on the cell membrane is very close,Cryptococcus cells by upregulated genes S100A10 causes cryptococcal capsular surface bound plasminogen is catalyzed more,to make them more easily activated,and S100A10 protein on the cell membrane plasminogen Cryptococcus on,the distance urokinase receptor close.When Cryptococcus invasive cells,studies have documented foreign Cryptococcus can without tissue plasminogen(tPA),when present in the cells of their own expression of urokinase,which urokinase urokinase receptor catalyzed by S100A10 nearby We had a large number of active plasminogen activator to plasmin,plasminogen and then destroy the extracellular matrix layer,cryptococcal through cell pathway through the blood-brain barrier more easily.In this study,the role of S100A10 were incubated and cryptococcal brain microvascular endothelial cells,and further research and demonstration.objectsCryptococcus can prove microvascular endothelial cells S100A10 gene regulated by the brain to activate the mouse urokinase-plasminogen system,thus cryptococcosis can more easily cross the blood.methods1.Setting the experimental group and the control group of mice with vascular endothelial cell bEnd.3 and Cryptococcus neoformans strains B3501 incubation,incubation time of 0h,2h,4h,8h,16 h,24h,perform Real-time Quantitative PCR and Western-blot S100A10 and urokinase were detected in mRNA expression and protein levels,respectively.2.Lentivirus plasmid,infection detection efficiency,and explore the best conditions bEnd.3 infected cells and by Real-time Quantitative PCR and Western-blot detected gene silencing S100A10,S100A10 urokinase gene and mRNA and gene in common protein expression levels.3.Transwell in vitro BBB model mice set silencing S100A10 after the experimental group did not S100A10 gene silencing bEnd.3 normal cells as the control group,respectively,and the wild-type strain B3501 incubation experiments,the amount of bacteria detected by the model.With a chromogenic substrate reagent S-2444 and S-2251 Detection of urokinase-related activation of plasminogen system.Results1.The longer the incubation time,S100A10 and UPA gene,the higher the amount of mRNA and protein levels.Wherein S100A10 at 2h,16 h,24h groups mRNA significant differences,t test,P <0.05;S100A10 protein in 24 h group differences,t test,P <0.05;UPA gene in 16 h,24h groups mRNA and control group significant differences,t-test,p <0.05;at 8h,16 h,24h were significantly different UPA protein group and the control group,the t-test,p <0.05.2.After building S100A10 gene lentivirus,found that cells infected bEnd.3 best rms and bEnd.3 normal cell morphology in a MOI of 20;after gene silencing S100A10,S100A10 gene and UPA gene mRNA and protein levels,respectively corresponding to the respective control groups decreased,and there are significant differences,t-test,P <0.053.Transwell model S100A10 normal and silent groups were constructed,the number of colonies by the model with extended incubation time,the overall trend is increasing.Especially the number of colonies in 8h group,16 h group,24 h group had significant difference by t-test p <0.05;at the same time the amount of C.neoformans is activated each group of urokinase and plasminogen also have significant differences,plasmin protein group 8h,16 h,24h three time points were significantly different,t examination P <0.05;urokinase protein group at 16 h,24h time two groups were significantly different by t test,P <0.05ConclusionCryptococcus S100A10 gene expression by endothelial cells up-regulated activation of urokinase-plasminogen system,its ability to promote through the blood brain barrier. |
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