平足蛋白（PDPN）在肝星状细胞活化以及肝纤维化中的作用分析.docx

肝纤维化及肝硬化DOI:10.12449/JCH240316平足蛋白(PDPN)在肝星状细胞活化以及肝纤维化中的作用分析三Q意。，杨广越”，张玮3浦幽,赵鑫”，马文婷小，刘旭凌邛，吴柳K,陶乐小,刘成Q上海中医药大学附属普陀医院a.肝病实验室，b.中心实验室，c.感染科，上海200062三三：文城,liucheng0082010163wm(ORCID:0000-0002-8741-6169)；陶乐，talent901036(g>163.m(ORCID:0000-0002-3659-8134)摘要：目的探究平足蛋白(PDPN)在肝星状细胞活化以及肝纤维化中的作用，并对其机制进行初步探讨.方法收集2019年9月2022年6月首次就诊于上海中医药大学附属普陀医院感染科的75例慢性乙型肝炎患者肝活检组织样本，实时定量PCR(RT-PCR)和免疫组化检测PDPN在不同肝纤维化分期患者肝组织中的表达.12只雄性C57/BL6小鼠随机分为正常组和模型组，每组各6只模型组腹腔注射含10%CCI4,对照组注射等体积橄榄;映6周，HE染色、天3B三红染色观察肝组织病理学变化，分离小鼠肝脏原代细胞，检测PDPNmRNA在各类型细胞中表达;使用PDPN蛋白处理小鼠原代肝星状细胞，并使用NF-KB抑制剂BAY11-7082处理,分析PDPN诱导肝星状细胞炎症因子表达情况o计量资料2组间骏采用成组1检验，多组间比较采用单因素方差分析，进一步两两比较采用LSD-检验。两组数据相关性分析采用SPearman相关性分析法结果慢性乙型肝炎患者肝活检样本中，PDPNmRNA在正常肝脏中表达较低,S3、S4期肝组织中表达显著升高,与正常肝组织比较差异均有统计学意义(P值均<0.001)；免疫组化染色结果显示，PDPN阳性表达主要集中在纤维间隔及肝窦，S4期肝组织PDPN口璀面积比SO期表达显著升高(f=8892,P=O.001)正常组小鼠中PDPN主要在肝嫁达少许,在Cd4侬小鼠中PDPN表班三高(f=0.95,P<0.001),主三三F维间隔趣；RT-PCR结,PDPNmRNA在CCL蹴小鼠中显著升高(f=11.25,P=Q.002).与肝细胞、肝星状细胞、Kupffer细胞和肝内胆管细胞相比，PDPN在肝要内皮细胞中明显高表达(F=20.56,P<0.001);PDPN蛋白如型小鼠原代肝星状细胞15min,婶相关因子TNF-a、CCL3、CXCL1和CXCR1的mRNA表达，与对照组相比均显著升高(尸值均<0.05),BAY11-7082(NF-KB信号抑制剂)处理后，上述炎跳标水平均明显下降(产值均<005)结论在肝纤维化中主要表达在肝窦内皮细胞的PDPN增加，其通过NF-KB信号调节肝星状细胞活化，促进肝纤维化进展。关键词：肝纤维化；肝星状细胞；肝实内皮细胞；平足蛋白基金项目：自行将基金(20ZR1450300)；国家自金(82004106)；区自主创惭项目(ptkwws202112,ptkwws202223)；上海市普陀区中心医院科研百人计划(2022-RCJC-02,2022-RCJC-04,2022-RCLH-01)；普陀杏林优青培养计划(ptxiyq2301)RoleofpodoplanininhepaticstellatecellactivationandliverfibrosisWANGZhiyPblYANGGUangy3,ZHANGWePlPUYaqiOn于,ZHAOXirrd,MAWentinc,LIUXUM及C,WULiifz,TAOL,c,LIUChery.(a.LaboratoryofLiverDiseases,b.CentralLaboratory,c.DepartmentOflnfectiousDiseases,PutuoHospitalAffiliatedtoShanghaiUniversityofTraditionalChineseMedicine,Shanghai200062,China)Correspondingauthors:LIUCheng,Hucheng0082010(ORCID:0000-0002-8741-6169);TAOLe,talet901036(8)(ORCID:0000002-3659-8134)Abstract:ObjectiveToinvestigatetheroleandmechanismofpodoplanin(PDPN)inhepaticstellatecell(HSC)activationandliverfibrosis.MethodsLiverbiopsysampleswerellectedfrom75patientswithchronichepatitisBwhoattendedDepartmentofInfectiousDiseases,PutuoHospitalAffiliatedtoShanghaiUniversityofTraditionalChineseMedicine,forthefirsttimefromSeptember2019toJune2022,andRT-PCRandimmunohisthemistrywereusedtomeasuretheexpressionofPDPNinlivertissueofpatientsindifferentstagesofliverfibrosis.Atotalof12maleC57BL6micewererandomlydividedintocontrolgroupandmodelgroup.Themiceinthemodelgroupweregivenintraperitonealejectionof10%Ca,andthoseintecontrolgroupwereinjectedwithanequalvolumeofoliveoil,for6weeks.HEstainingandSiriusRedstainingwereusedtoobserveliverhistopathologicalchanges;primarymouselivercellswereseparatedtomeasurethemRNAexpressionofPDPNinvarioustypesofcells;primarymouseHSCsweretreatedwithPDPNprotein,followedbytreatmentwiththeNF-BinhibitorBAY11-7O8,tomeasuretheexpressionfinflammato<yfectorsinHSCsinducedbyPDPN.Teindependent-samplesftestwasusedforcomparisonofnonellydistributedcontinuousdatabetweentwogroups;aone-wayanalysisofvariancewasusedforcomparisbetweenmultiplegrps,andtheleastsignificantdifferenceMeStwasusedforfurthercomparisbetweentwogroups.TeSpearmancorrelationanalysiswasusedtoinvestigatedatacorrelation.ResuteAsfortheliverbiopsysamples,therewasarelativelylowmRNAexpnessilevelofPDPNinnormalliver,andtherewasasignificantineaseinthemRNAexpressilevelofPDPNinlivertissueofstageS3orS4fibrosis(allP<0.001).ImmUnohistOChemiCalstainingshelvedthatPDPNwasmainlyexpressedinthefibrousseptumandthehepaticsinusoid,andthePDPNpositiveareainS4livertissuewassignificantlyhigherthanthatinSOlivertissue(f=8,892,P=0,001).Innormalmice,PDPNwasmainlyexpressedinthehepaticsinusoid,andtherewasasignificantincreaseintheexpressionofPDPNinCamodelmice(占0.95,P<Q001),mainlyinthefibrousseptum.RT-PCRshowedasignificantincreaseinthemRNAexpressionofPDPNintheCQ1modelmice(UlL25,P=Oj002).Comparedwithhepatocytes,HSCs,Kupffercells,andbileductendothelialcells,hepaticsinusoidalendothelialcellsshewedasignificantlyhighexpressionlevelofPDPN(F=2056,P<0,001).Comparedwiththecontrolgroup,theprimaymouseHSCstreatedbyPDPNproteinfor15minutesshowedsignificantineasesinthemRNAexpressionlevelsoftheinflammati-relatedfactorsTNFaiCCL3,CXCLl,andCXCRl(allP<0.05),andthereweresignificantreductionsinthelevelsoftheseindicatorsaftertreatmentwithBAY11-7O82(allP<0,05).ConclusionThereisanineaseintheexpressionofPDPNmainlyinhepaticsinusoidalendothelialcellsduringIiverfibrosis,andPDPNregulatesHSCactivationandpromotestheprogressionOfliverfibrosisviatheNF-Bsignalingpathway.Keywords:HepaticFibrosis;HepaticStellateCell;HepaticSinusEndothelialCell;PodoplaninResearchfunding:ShanghaiIMaturaISdenceFOUndatiOn(20ZR1450300);NationalNaturalScienceFoundatbnofChina(82004106);IndenpentInnovationofShanghaiPutouDistrictProject(ptkvwvs202112,ptkwws202223);Projectof,'100ProfessionalsTrainingPlan,ofShanghaiPutDistrictCentralHospital(2022-ROC-02,2022-RCJC-04,2022-RCLH-01);PutuoXinglinExcellentYouthTrainingProgram(ptxlyq2301)肝纤维化是由多种病因引起的慢性、进行性、弥漫性肝病，其主要病理特征是肝内细胞外基质过度增生与异常沉积所导致的肝脏结构和