hiv-1感染孕妇胎盘形态免疫学变化及hiv-1宫内传播机制研究

HIV1感染孕妇胎盘形态免疫学变化及HIV1宫内传播机制研究HIV1HIV1摘要目的方法HHHIIIVVV111感染孕妇胎盘形态免疫学变化及HHHIIIVVV111宫内传播机制研究博士研究生董晓梅指导教师王自能教授摘摘摘要要要目的目的目的1了解HIV1感染孕妇胎盘的超微结构变化,探索病毒是否可以感染胎盘及其在胎盘中的定位,对比分析抗逆转录药物治疗对病毒感染胎盘的影响2分析HIV1感染对孕妇胎盘中T淋巴细胞和巨噬细胞数量变化的影响,及其对母胎界面的TH1/TH2相关细胞因子水平的影响3明确CD4、CCR5、CXCR4及DCSIGN分子在晚孕胎盘和早孕绒毛的存在及表达情况4探索HIV1以何种方式感染滋养细胞,病毒在滋养细胞中是否存在复制及CCR5、CXCR4和DCSIGN在病毒入胞过程中的作用。方法方法方法1收集11例HIV1感染孕妇胎盘为病例组,13例正常孕妇胎盘为对照组,进行HIV1P24抗原的免疫组化检测和激光扫描共聚焦显微镜LSCM观察选择其中的4例HIV1感染孕妇胎盘和2例正常孕妇胎盘进行透射电子显微镜TEM观察。2免疫组化检测病例组与对照组孕妇胎盘中CD3阳性细胞、CD68阳性细胞数量及IL2、IL4、TNFA表达水平。3增加10例早孕流产绒毛为第3组,免疫组化检测并比较3组孕妇胎盘或绒毛组织中HIV1相关受体CD4、CCR5、CXCR4和DCSIGN分子的存在情况及其表达强度。结果暨南大学博士学位论文中文摘要4利用人绒毛膜癌JEG3细胞株和BEWO细胞株分别与游离病毒和感染有HIV1IIIB的C8166T细胞共培养,通过PCR检测细胞基因组DNA中是否有HIV1GAG基因的整合,以验证病毒进入滋养细胞的可能性应用ELISA法检侧培养上清中的HIV1P24抗原和RTPCR法测定病毒载量,以证实是否有子代病毒的释放。流式细胞仪检测人绒毛膜癌JEG3和BEWO细胞株表面CCR5、CXCR4和DCSIGN分子的存在情况,利用抗体阻断实验分析它们在病毒入胞机制过程中的作用。结果结果结果1两组胎盘标本在普通显微镜下均未发现HIV1P24抗原的免疫组化阳性表达LSCM发现病毒定位于滋养细胞、间质细胞和胎儿血管内皮细胞TEM发现,1例未接受抗病毒药物治疗的HIV1感染孕妇胎盘滋养细胞内有约100NM大小的病毒样颗粒结构存在,可见病毒进入细胞的吞噬小泡,病毒样颗粒周围溶酶体聚集,细胞有内质网扩张和空泡样改变,间质HOFBAUER细胞增多。其余阳性胎盘TEM下没有发现类似病毒颗粒与超微结构的改变。2病例组胎盘中的CD3、CD68阳性细胞数均高于对照组P0001,200倍视野下两组的CD3、CD68阳性细胞分别为3611VS1006和20527VS12234IL2在滋养细胞层和绒毛间质弱表达,两组间差异无统计学意义病例组IL4的表达低于对照组P005,两组的平均PU值为251075VS322071TNFA的表达则高于对照组P005,两组的平均PU值为386108VS301086。3CD4在早孕绒毛和晚孕胎盘中的表达存在个体差异,早孕组阳性率为707/10,晚孕对照组为61548/13,病例组为72738/11,三组的阳性率差异无统计学意义。CXCR4、CCR5及DCSIGN分子在胎盘均有表达,三者均定位于滋养细胞和绒毛间质细胞,且在早孕绒毛中的表达强度低于晚孕对照组和病例组,差异有统计学意义。4通过PCR扩增滋养细胞中HIV1GAG基因的整合、ELISA检测HIV1P24抗原和病毒载量测定发现,游离病毒不易感染滋养细胞系与被感染的T细胞共培养,HIV1IIIB可以进入并整合到滋养细胞遗传物质中感染后培养48小时检II结论暨南大学博士学位论文中文摘要测到子代病毒的复制与释放,72小时呈增加趋势滋养细胞系低水平表达CCR5、CXCR4和DCSIGN,但抗体阻断实验不能阻止HIV1对滋养细胞的感染。结论结论结论1本研究从组织学形态层面证实,在HIV1感染孕妇,病毒可以感染胎盘的滋养细胞,且胎盘的超微结构有所改变病毒在胎盘中主要定位于滋养细胞、胎盘的间质细胞和胎儿血管内皮细胞。但是,在接受抗病毒药物治疗的HIV1感染孕妇胎盘中没有发现类似病毒颗粒与超微结构的改变,提示产前干预在阻断HIV1感染垂直传播可能具有重要意义。2胎盘受到HIV1感染时,细胞免疫可能有所增强IL4表达降低,TNFA表达增加,提示HIV1的感染可能促使母胎界面向免疫排斥转变,对妊娠结局可能造成不利影响。3胎盘组织中存在HIV1各种受体分子,具备感染HIV1的分子基础。胎盘中CD4分子的表达存在个体差异CCR5、CXCR4及DCSIGN分子在晚孕胎盘中的表达较高,可能是HIV1感染宫内传播多发生于孕晚期的原因之一。4滋养细胞系不易被游离的HIV1感染,与被感染的T细胞系共培养可以提高滋养细胞易感性。病毒在滋养细胞中存在复制。滋养细胞系表面低水平表达的CCR5、CXCR4和DCSIGN可能与病毒进入滋养细胞的机制无关。关键词HIV1胎盘LSCMTEMCD3CD68IL2IL4TNFACD4CCR5CXCR4DCSIGN滋养细胞JEG3/BEWOIIIMORPHOLOGICALANDIMMUNOLOGICALCHANGESOFTHEPLACENTAFROMHIV1SEROPOSITIVEWOMENANDINTRAUTERINETRANSMISSIONMECHANISMOFHIV1INFECTIONABSTACTOBJECTIVEMETHODS暨南大学博士学位论文英文摘要MORPHOLOGICALANDIMMUNOLOGICALCHANGESMMOORRPPHHOOLLOOGGIICCAALLAANNDDIIMMMMUUNNOOLLOOGGIICCAALLCCHHAANNGGEESSOOOFFFTTTHHHEEEPPPLLLAAACCCEEENNNTTTAAAFFFRRROOOMMMHHHIIIVVV111SSSEEERRROOOPPPOOOSSSIIITTTIIIVVVEEEWWWOOOMMMEEENNNAAANNNDDDINTRAUTERINETRANSMISSIONMECHANISMOFHIV1INFECTIONIINNTTRRAAUUTTEERRIINNEETTRRAANNSSMMIISSSSIIOONNMMEECCHHAANNIISSMMOOFFHHIIVV11IINNFFEECCTTIIOONNDOCTORCANDIDATEDONGXIAOMEISUPERVISORPROFWANGZINENGABSTACTAABBSSTTAACCTTOBJECTIVEOOBBJJEECCTTIIVVEE1TOUNDERSTANDTHEULTRASTRUCTURALCHANGESOFPLACENTAFROMHIVSEROPOSITIVEWOMENANDCLARIFYTHELOCATIONOFVIRUS,COMPARATIVEANALYSISOFTHERELATIONSHIPBETWEENHARRTTREATMENTWITHTHESTATUSOFHIVINFECTIONOFPLACENTAS2TOANALYSISTHEEFFECTOFHIV1INFECTIONONTHECHANGESOFNUMBEROFIMMUNECELLSINPLACENTAANDTHELEVELOFRELATEDCYTOKINESONMATERNALFETALINTERFACE3TOCLARIFYTHEEXPRESSIONOFCD4,CCR5,CXCR4ANDDCSIGNINTHEPLACENTA/CHORIONICVILLI4TOEXPLORETHEMECHANISMSOFHIV1ENTERTROPHOBLASTCELLSANDTHEROLEOFCCR5,CXCR4ANDDCSIGNUNDERSTANDWHETHERTHEREISAREPLICATIONOFTHEHIV1INTROPHOBLASTCELLSMMMEEETTTHHHOOODDDSSS111PLACENTAESFROMHIV1SEROPOSITIVEWOMENAND13PLACENTAESFROMNORMALPLACENTASWERECOLLECTEDTRANSMISSIONELECTRONMICROSCOPYTEM,HIV1P24ANTIGENIMMUNOHISTOCHEMICALDETECTIONANDANDLASERSCANNINGCONFOCALMICROSCOPELSCMDETECTIONWEREDONEVRESULTS暨南大学博士学位论文英文摘要2THEEXPRESSIONOFCD3,CD68,IL2,IL4,ANDTNFAINTHEPLACENTA/CHORIONICVILLIWEREDETECTEDBYTHEIMMUNOHISTOCHEMISTRY3ADDITIONALCOLLECTIONOF10CASESOFEARLYPREGNANCYABORTIONVILLIASGROUP3,THEEXPRESSIONOFCD4,CCR5,CXCR4ANDDCSIGNINTHEPLACENTA/CHORIONICVILLIWEREDETECTEDBYTHEIMMUNOHISTOCHEMISTRY4JEG3/BEWOCELLSWERECOINCUBATEDWITHFREEVIRUSHIV1IIIBORINFECTEDTCELLC8166/HIV1IIIB,PCRAMPLIFINGHIV1GAGGENEWASUSEDTODETECTWHETHERHIV1CANINFECTTROPHOBLASTICCELLELISATESTINGHIV1P24ANTIGENWASUSEDTODETECTWHETHERTHEREISAREPLICATIONOFHIV1INTROPHOBLASTCELLSTHEMOLECULEOFCCR5,CXCR4ANDDCSIGNONTHECELLSURFACEWEREDETECTEDBYFLOWCYTOMETRY,BLOCKINGANTIBODIESTESTWEREUSEDTOANALYSISTHEROLEOFTHESEMOLECULESRESULTSRREESSUULLTTSS1THEREWASNOPOSITIVEEXPRESSIONOFTHEHIV1P24ANTIGENINTWOGROUPSPLACENTAESVIRUSWASFOUNDINTHETROPHOBLASTCELLS,STROMALCELLSANDENDOTHELIUMOFFETALCAPILLARSBYLSCMUNDERTHEVISUALFIELDOFTEM,VIRUSLIKEPARTICLESWEREFOUNDINTROPHOBLASTCELLSWITHABOUT100NMSIZECHANGESWEREFOUNDINTROPHOBLASTCELLSSUCHASEREXPANSION,VACUOLARDEGENERATION,ANDHOFBAUERCELLSINCREASEDINTHEPLACENTATHEREMAININGPOSITIVEPLACENTAESFOUNDNOSIMILARVIRUSPARTICLESANDULTRASTRUCTURALCHANGES2THENUMBEROFCD3ANDCD68POSITIVECELLSINCASEGROUPWASHIGHERTHANTHECONTROLUNDERTHEVISUALFIELDWITH200TIMESMAGNIFICATION,THENUMBEROFCD3ANDCD68POSITIVECELLSOFGROUPSOFCASEANDCONTROLWERE3611VS1006AND20527VS12234,RESPECTIVELYIL2WASWEAKLYEXPRESSEDATTROPHOBLASTCELLSANDSTROMALOFPLACENTA,THEREWASNOSIGNIFICANTDIFFERENCEBETWEENTHETWOGROUPSTHEEXPRESSIONLEVELOFIL4OFCASEGROUPWASLOWERTHANTHECONTROL,THEAVERAGEPUVALUEOFTHETWOGROUPSIS251075VS322071,BUTTHEEXPRESSIONLEVELOFTNFAWASHIGHER,THEAVERAGEPUVALUEIS386108VS301086,RESPECTIVELYP005VICONCLUSION暨南大学博士学位论文英文摘要3THEREAREINDIVIDUALDIFFERENCESOFCD4EXPRESSIONINPLACENTA,THEPOSITIVERATEOFTHETHREEGROUPSWAS70,6154AND7273,ANDRESPECTIVELYTHEREWASNOSTATISTICALDIFFERENCEAMONGTHREEGROUPSALLOFCXCR4,CCR5,ANDDCSIGNHAVEPOSITIVEEXPRESSIONINTHEPLACENTA,THEYALLLOCATEDATTHETROPHOBLASTCELLSANDSTROMALOFVILLITHELEVELOFCXCR4,CCR5,ANDDCSIGNEXPRESSIONINCHORIONICVILLIFROMFIRSTTRIMESTERWERELOWERTHANTHEMINPLACENTASFROMTHETHIRDTRIMESTER,THEDIFFERENCEAMONGTHETHREEGROUPSWASSIGNIFICANT4TROPHOBLASTICCELLLINESWERENOTSUSCEPTIBLEOFTHEFREEHIV1ASDETECTEDBYPCRANDRELEASEOFP24ANTIGENINCONTRAST,HIV1IIIBWASFOUNDINTEGRATINGINTOTROPHOBLASTICCELLDNAWHENCOINCUBATEDWITHINFECTEDTCELLAFTER48HOURS,PROGENYVIRUSPARTICLESWEREDETECTEDINTHESUPERNATANT,AFTER72HOURS,THERELEASEOFPROGENYVIRUSPARTICLESINCREASEDTROPHOBLASTICCELLLINESEXPRESSEDLOWLEVELSOFCHEMOKINERECEPTORSCCR5,CXCR4ANDDCSIGNTOTESTIFTHOSEMOLECULESWEREUSEDASRECEPTORSFORHIV1INFECTION,PLACENTALCELLSWEREPRETREATEDWITHANTIBODIESTOCCR5,CXCR4ANDDCSIGNNONEOFTHESETREATMENTSINHIBITEDHIV1INFECTIONCONCLUSIONCCOONNCCLLUUSSIIOONN1THISSTUDYCONFIRMEDFROMTHEMORPHOLOGYTHATHIV1CANINFECTTHETROPHOBLASTCELLOFPLACENTAANDSOMECHANGESOCCURREDINULTRASTRUCTUREOFTHEPLACENTATHEVIRUSPARTICLESMAINLYLOCATEDATTROPHOBLASTCELLS,STROMALCELLSANDENDOTHELIUMOFFETALCAPILLARSTHEREWERENOSIMILARFINDINGSOFTHEVIRUSPARTICLESANDULTRASTRUCTURALCHANGESAMONGPLACENTAESFROMWOMENWHORECEIVEDHARRTTREATMENT2WHENPLACENTAINFECTEDBYHIV1,CELLIMMUNITYMAYBEENHANCEDIL4EXPRESSIONDECREASEDANDTNFAEXPRESSIONUPREGULATED,SUGGESTINGTHATHIV1INFECTIONMAYPROMPTTHEMATERNALFETALINTERFACECHANGESTOTHEIMMUNEREJECTIONTHISMAYHAVEANADVERSEIMPACTONTHEOUTCOMEOFPREGNANCY3WITHTHEEXPRESSIONOFALLTHECORECEPTORSANDRELATEDMOLECULESOFHIV1,PLACENTAPOSSESSEDTHEMOLECULARBASISOFHIV1INFECTIONTHEREAREINDIVIDUALVIIKEYWORDS暨南大学博士学位论文英文摘要DIFFERENCESINTHEEXPRESSIONOFCD4MOLECULESINTHEPLACENTATHEEXPRESSIONOFCCR5,CXCR4ANDDCSIGNMOLECULESINTHEPLACENTASFROMTHETHIRDTRIMESTERWEREHIGHERTHANCHORIONICVILLIFROMFIRSTTRIMESTER,WHICHMAYBERELATEDWITHTHEFACTTHATMOSTOFMTCTOCCUREDATTHETHIRDTRIMESTERSTAGE4TROPHOBLASTCELLLINESWERENOTSUSCEPTIBLETOFREEHIV1,BUTCOINCUBATEDWITHTHEINFECTEDTCELLCANINCREASETHESUSCEPTIBILITYOFTROPHOBLASTCELLSTHEVIRUSCANREPLICATEINTHETROPHOBLASTCELLTROPHOBLASTCELLLINESEXPRESSEDLOWLEVELSOFCCR5,CXCR4ANDDCSIGN,BUTTHEYMAYINDEPENDENTOFTHEENTERINGMECHANISMOFHIV1KKKEEEYYYWWWOOORRRDDDSSSHIV1PLACENTALSCMTEMCD3CD68IL2IL4TNFACD4CCR5CXCR4DCSIGNTROPHOBLASTCELLSJEG3/BEWOCELLSVIII目录中文摘要英文摘要目录前言11HIV1病原学特征与HIV/AIDS的流行状况12HIV1的母婴传播63HIV1宫内传播机制的研究进展134课题设计思想和意义17参考文献18第一章HIV1感染孕妇胎盘中病毒感染状况及超微结构变化251材料和方法262结果313讨论32参考文献35第二章HIV1感染孕妇胎盘中免疫细胞及TH1/TH2的变化411材料和方法422结果4