C1抑制剂在血管通透性调节和抑制白细胞内皮迁移的作用课件.ppt

1、2020年10月2日1 A serpin(serine proteinase inhibitor).Serpins inactivate proteases by forming a stable complex with the protease.Protease recognizes a substrate-like region of the inhibitor that is referred to as the reactive center loop.C1 INHIBITOR FUNCTION2020年10月2日2ProteaseSerpinReactive centerThe p

2、rotease recognizesthe reactive center of theinhibitor.Cleavage of thereactive center loop isfollowed by:(1)covalent bond formaton between the protease active site serine and the reactive center amino acid of the serpin.(2)rearrangement of the serpin with insertion of the reactive center loop(in yell

3、ow)into sheet A(in red),which results in movement of the protease from one pole of the molecule to the other.This results in structural distortion of the protease.2020年10月2日32020年10月2日4C4b2a3bC3bBb3bC5C5aC5b+C6,C7,C8,C9C5b-9Membrane Attack ComplexCLASSICAL PATHWAYAntigen-antibodycomplexmicrobeMBL+MA

4、SP 1,2LECTIN PATHWAYALTERNATIVE PATHWAYmicrobe+C3b,B,D,PC3b,Bb,PC1q,C1r,C1sC4C2C4b2aC3C3aC3b+C4b2aC3b+C3bBbPC1INHC1INHFactor IFactor HC1INH2020年10月2日5 Endothelial cell prolylcarboxypeptidaseFactor XIINegativelychargedsurfaceXIIaC1INHPrekallikreinHigh molecularweight kininogen KallikreinHigh molecula

5、rweight kininogenBradykininC1INHC1INHContact System Activation2020年10月2日62020年10月2日72020年10月2日8Pathophysiology of Hereditary Angioedema Because there is clearcut evidence for both complement and contact system activation,the question arises as to which is responsible for generation of the mediator o

6、f angioedema.Until recently,some data suggested that the mediator was a product of complement system activation while other data suggested that it was derived from contact system activation.2020年10月2日9 Define mediator(s)of angioedema Test new therapies and define their mechanism of action Pathophysi

7、ology of vascular permeability Analysis of variability of symptoms in HAEC1 Inhibitor Knockout Mouse.Why?2020年10月2日102020年10月2日11Wild TypeC1INH+/-2020年10月2日12C1INH genotype +/+/+-/-/-Evans blue dye -+C1INH treatment -+Does intravenous C1INH reverse theincreased vascular permeability in the C1INH kno

8、ckout mice?2020年10月2日13C1INH genotype+/+/-/-+/-/-C1INH treatment 0 0 0 +Mean .10 .15 .16 .11 .10 S.E.005 .008 .012 .003 .006 Vascular Permeability-Footpads2020年10月2日14C1INH genotype +/+-/-/-Bk2R genotype +/+/+-/-Evans blue dye +Is Angioedema Mediated by Bradykinin?Bradykinin induces increased vascul

9、ar permeability via the Bk type 2 receptor(Bk2R).C1INH-/-mice were mated with Bk2R-/-mice.Hypothesis:C1INH-/-,Bk2R-/-mice will be resistant to the development of increased vascular permeability.2020年10月2日15C1INH genotype+/+/-/-/-Bk2R genotype+/+/+/+-/-Treatment 0 0 0 0 Mean .10 .15 .16 .10 S.E.005 .

10、008 .012 .003 Vascular Permeability-Footpads2020年10月2日16C1INH genotype +/+-/-/-/-/-Treatment 0 0 Captopril Bk1RA Hoe140 Mean .2577 .4087 .8860 .4477 .0252Standard error .0284 .0806 .0773 .0667 .0252Intestinal Vascular Permeability2020年10月2日17 In addition,the increased vascular permeability in C1INH-

11、/-mice was reversed by treatment with the following:A recombinant variant Kunitz domain inhibitor,DX88(Dyax),that is highly specific for plasma kallikrein.A recombinant C1INH Ala443 Val,that inhibits the contact system but not the complement system.A Bk2R antagonist,Hoe140(Icatibant).These data all

12、support the hypothesis that the mediator of vascular permeability in C1INH deficiency is bradykinin.2020年10月2日182020年10月2日192020年10月2日20C1 Inhibitor Expresses Sialyl LewisxHECA452:monoclonal anti-sLewx and sLewaLanes 1-4:C1INH 8,4,2,1 gLane 5:U937 lysateLane 6:LEC11 lysateLane 7:CHO-K1 lysateLane 8:

13、BSACSLEX-1:monoclonal anti-sLewxLane 1:BSALanes 2-3:C1INH 5&2.5 gLane 4:U937 lysateDeglycosylation experiments:sLewx isexpressed on N-linked carbohydrate.2020年10月2日21Recombinant C1INH Expressed in LEC11 Cells Expresses the Sialyl Lewisx EpitopeLEC 11 cells express a(1,3)-fucosyltransferaseCHOK1 cell

14、s do not2020年10月2日22Endothelial Cell E and P SelectinCo-precipitate with C1 InhibitorHUVEC treated with TNF-a&H2O2,then incubated with C1 inhibitor.Cells lysed&immunoprecipitated with rabbit anti-C1 inhibitor antiserum.SDS-PAGE,Western blot probed with antiserum to E or P selectin.E SelectinP select

15、inBinding of C1INH to CHO cells thatexpress either E or P selectin alsowas demonstrated by FACS.2020年10月2日23C1INH Suppresses U937 Cell Adhesion to Endothelial CellsHUVEC monolayers treated with TNF-a&H2O2,then incubated with or withoutC1INH(31.5 500 g/ml).BCECF-AM labelled U937 cells added&incubated

16、 for60 min at 37oC,then washed&bound cells quantitated by analysis of fluorescenceintensity.2020年10月2日24HUVEC(3x104)were seeded onto an insert and grown in 24-well plates for 4 days,then stimulated with TNF alpha(50 ng/ml,18 h).The integrity of the monolayer was tested with FITC-BSA.U937 cells were

17、labeled with BCECF-AM,5x105 cells(100l)in the absence or presence of either native or reactive center cleaved C1INH at the indicated concentrations were incubated with the HUVEC at 37oC for 45 min with 600l medium in the lower chamber.The fluorescence intensity of 100 l medium from the lower chamber

18、 of each well was measured.Transwell050100150200250300350062.5125250500native C1INH ug/mlFluorescence Unit.Transwell050100150200250300062.5125250500Cleaved C1INH(ug/ml)Fluorescence UnitC1INH Inhibits U937 Cell Transmigration Across the Endothelium2020年10月2日25C1INH Blocks Carcinoembryonic AntigenBind

19、ing to E-selectin2020年10月2日26C1INH Blocks HL-60 Cells from Rolling on E-and P-Selectin Under Flow Conditions In VitroImmobilizedE-selectinCHO-PCells2020年10月2日27C1INH Inhibits TNF-a Induced Leukocyte Rolling In VivoC1INHiC1INHdN-C1INHPBS2020年10月2日28C1INH Blocks Leukocyte Infiltration inThioglycollate Induced Peritonitis2020年10月2日292020年10月2日302020年10月2日31演讲完毕，谢谢观看！Thank you for reading!In order to facilitate learning and use,the content of this document can be modified,adjusted and printed at will after downloading.Welcome to download!汇报人：XXX 汇报日期：20XX年10月10日32