幽门螺杆菌空泡毒素VACA抑制宿主免疫应答的机制研究

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1、上海交通大学硕士学位论文幽门螺杆菌空泡毒素（VacA）抑制宿主免疫应答的机制研究姓名：陶晶申请学位级别：硕士专业：细胞微生物学指导教师：刘晶星200704012+以上实验说明VacA可以抑制单核巨噬细胞Ca、ROS和TNF-α的表达，可能在H.pylori逃避宿主的免疫应答过程中起到重要的作用。为进一步研究VacA是否通过抑制ILK途径影响eNOS、ROS的表达，我们采用pcDNA3.1(+)质粒，构建含有ILK点突变序列的质粒（ILK蛋白359位氨基酸由谷氨酸变为赖氨酸），并电转入U937细胞，以筛选ILK蛋白活性减低的稳定表达细胞株，为下一步工作打下基础。关键

2、词幽门螺杆菌，空泡毒素，整合素连接激酶，反应性活性氧，2+Ca，TNF-αIIRESEARCHONMECHANISMOFH.PYLORIINHIBITINGHOSTIMMUNITYBYVACAABSTRACTHelicobacterpylori（H.pylori）isaGram-negativemicroaerophilicbacterium.Thebacteriahavebeenassociatedwithchronicgastritis,pepticulcer,gastricadenocarcinoma,andgastricmucosa-associatedl

3、ymphoidtissuelymphoma(MALT).H.pylorihasbeenclassifiedasaclassIcancerogenicagentbytheWorldHealthOrganization.H.pyloricanproducemanykindsoftoxinandenzyme,whichcanresistgastricacid,damagegastricmucosa,andcausegastroduodenalinflammation.Vacuolatingcytotoxin(VacA)isacytotoxinsecretedfromH.

4、pylori.Inadditiontoinducingvacuolationinmanykindsofcells,VacAcouldinducemitochondrialdamageandapoptosis,orinhibittheactivationandproliferationofTlymphocytes.Inthepreviousresearch,wefoundthatVacAcouldinhibittheintergrin-linkedkinase(ILK)andendothelialnitric-oxidesynthase(eNOS)atmRNAlev

5、el.InordertostudythemechanismsofVacAinhibitingthehostimmuneresponse,weusedisogenicH.pylori(thewildtypeH.pyloriNCTC11638andvacA-deficientH.pylorimutantstrain11638-ΔvacA)tostimulatemonocyte/macrophage-likeU937cells.Weusedconfocallaser-scanningIII2+microscopytodetectetheproductionofCaand

6、reactiveoxygenspecies(ROS)inU937cellsindifferenttimeandfoundthat11638-ΔvacAcould2+stimulatemoreCaandROSinU937cellsthantheNCTC11638after35hinfection(P<0.01).WecollectedthesupernatantsofH.pyloriandU937cellstodetectTNF-αlevelandfoundthatbothH.pyloricanstimulatetheTNF-αinU937cells.But1163

7、8-ΔvacAcanstimulatemoreTNF-αthanNCTC11638(P<0.01).TheseresultssuggestedthatVacAcouldinhibitthe2+productionofCa,ROSandTNF-αinmonocyte/macrophage-likeU937cells,helpingH.pyloriescapehostimmuneresponse.InordertoconfirmourhypothesisthatVacAmightinfluenceROSexpressionbyinhitingILK,weconstru

8、ctedt