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INTRODUCTIONPatientswithchronicHBVinfectionshowimmunetolerance,whichhasbeenattributedtothepoorTcellresponsesintheperipheralblood慢性乙型肝炎的免疫难受机制主要是由于T细胞应答不足FewstudieshaveinvestigatedifdifferentiationofTcellsfromHSCsisassociatedwithaweakTcellresponseinchronicHBVinfection.很少有研究探讨慢性乙型肝炎的T细胞应答不足是否与造血干细胞分化异常有关SomestudiesreportedthatbonemarrowtransplantationledtotheablationofpersistenthepatitisB一些研究报道慢乙肝病人骨髓移植后得到治愈
1TcellsarederivedfromCD34+BMcells,healthyTcellsshouldbecontinuallygeneratedfromCD34+BMHSCsinchronicHBVinfectionpatient，however,thereislittlereconstitutionwithTcellregenerationfromtheHSCsT细胞来源于CD34+骨髓造血干细胞，在慢乙肝的病人体内造血干细胞应不断新生健康T细胞来达到免疫重建，然而病人这种现象却很少见。WehypothesizedthatCD34+BMHSCscouldbeinfectedwithHBVDNA,evenwhichcouldcausedeficientTcelldifferentiationanddevelopmentsothisstudywasperformed我们推断CD34+骨髓造血干细胞可能感染了乙肝病毒，从而使造血干细胞分化T细胞出现异常，因此我们设计了该实验
2PATIENT8patientsnewlydiagnosedwithpersistentHBVinfectionmorethanoneyearandthe7healthyindividualswerealsoenrolled8例诊断为慢性乙肝病毒感染一年上的病人和7例健康人入组Theinclusioncriteria:adiagnosisofHBVDNA≥105copiesml-1,HBsAg,anti-HBcpositive,anti-HBsnegativeandnohistoryofantivirustreatment.入组条件：HBVDNA≥105copiesml-1HBsAg,anti-HBc阳性,anti-HBs阴性，没有抗病毒史Patientswereexcluded:iftheyhadothermixedcausesofchronicliverdiseasesuchashepatitisC,alcoholabuseorautoimmuneliverdisease,oriftheyhadadiagnosisoflivercirrhosisandhypersplenia除外条件：合并其他肝病如丙肝，酒精肝，自身免疫性肝病以及肝硬化，脾功亢进。
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5measurementofHBSgenemRNAfromCD34+BMHSCsofpatientsandcontrolbyRT-PCR慢乙肝病人和健康人的造血干细胞的HBS基因的mRNA检测TheHBSgeneprimers(HBS基因引物):forward5'-TATCGCTGGATGTGTCTGC-3';reverse,5'-AGACTTGGCCCCCAATACC-3GAPDHprimers(内参基因引物):forward5-TGCACCACCAACTGCTTAGC-3reverse5-GGCATGGACTGTGGTCATGAG-3totalRNAwasextracted总RNA的提取reversetranscription逆转录ThegenewereamplifiedusinganRTreaction逆转录产品的扩增Electrophoresisagarosegel.琼脂糖凝胶电泳
6Treegroupsweredesigned:HSCsofthepatientsandthehealthydonorsasnegativecontrolsandHepG22.15cellsasthepositivecontrolsforFISH三组细胞：慢乙肝病人组造血干细胞，健康人造血干细胞（阴性对照），HepG22.15细胞（阳性对照）进行原位荧光杂交检测Treegroupofcells三组细胞.Treatedbykcl用kcl处理Treatedbykclandcochicine用kcl和秋水仙碱处理chromosomalsamples染色体标本Nucleisamples.细胞核标本denatureandwereincubatedforwithHBVprobe标本变性并与HBV探针杂交冰醋酸固定FITClableHBVprobeandcounterstainedDAPIFITC标记HBV探针和DAPI复染imageswereacquired.byfluorescentmicroscope荧光显微镜摄片
7CD34+BMHSCsandOP9-DL1coculture(造血干细胞与OP9-DL1细胞共培养)OP9-DL1cellswereobtainedfromDr.J.C.ZunigaPflucker(UniversityofToronto)(细胞株来源于多伦多大学Pflucker教授）OP9-DL1cellswasseeded24hbeforeOP9-DL1提前24小时接种CD34+HSCscocultureontomonolayerofOP9-DL1cellsforgenerateTcellfor25day造血干细胞接种于OP9-DL1基质细胞上进行共培养25天诱导T细胞Pleasenote!请注意！1.AddmediumIMDMwith20%FCS,Flt3L,IL-7,SCF2.Every4–5transferfreshOP9-DL1andmedium1.加含20%胎牛血清的IMDM培基2.4-5天换液，更换OP9-DL1细胞HealthyHSCsandOP9-DL1coculturesPatientHSCsandOP9-DL1cocultures
8TcellexpansionanddetectionofTcellphenotype收集诱导的T细胞进行表型检测TcellsfromOP9-DL1cocultureofpatientsandcontrols从OP9-DL1共培养物中收集诱导的T细胞labelandstainwiththeantibodyindark在暗室内标记相应抗体determinethepercentageofpositivecellsbyFACSondays14andday25第14和25天时进行流式细胞仪检测阳性细胞的百分数APC-CD3,PE-TCRaβ,FITC-CD4,PE-CD8,PE-CD25Viablecellsweregatedbasedonthelymphocytegate目标T细胞选择淋巴细胞射门
9TcellsproliferationandcytokineproductionassaysT细胞增殖度和细胞因子检测CFSEisafluorescentdyeusedtolabelcellswhichdilutionwasmeasuredbyflowcytometryforassessingT-cellsproliferationCFSE是一种标记活细胞的荧光染料，通过流式细胞仪检测标记细胞荧光下降的强度来检测细胞增殖度TheCDIistheratiooffluorescencedilutionofthePHA-stimulatedtononPHA-stimulatedcells,whichwasusedtocalculatecellproliferationdynamicsCDI是增殖指数，用PHA刺激后标记细胞荧光下降的强度比未用PHA刺激的标记细胞荧光下降的强度
10TcellsfromOP9-DL1coculturesonday25wereincubatedwithCFSE将T细胞用CFSE标记Non-PHAculturefor72h无PHA培养72小时AddPHAculturefor72h加PHA培养72小时thepercentageoffluorescencedilutionwasanalyzedbyFACS用流式细胞仪检测细胞荧光下降的百分数supernatantswerecollectedforanalysisofIL2andIFN-γlevelsusingspecificELISAkits收集上清液用ELISA检测IL2和IFN-γ水平
11RESULTSRT-PCRThe403bpRT-PCRproductscorrespondingtotheamplifiedHBSgenefragmentswereobservedinCD34+HSCsfromthepatientsbyagarosegelelectrophoresisbutnoexpressionofthesegenefragmentswasobservedintheHSCsfromthehealthycontrols通过RT-PCR检测发现：403bpHBS基因片段在慢乙肝病人的CD34+HSCs中表达，而正常对照组无该基因表达LaneM,DNAmarker(DL2000);lane1,healthycontrollane2,HepatitisB(HB)Sgene(403bp),lane3,internalcontrolgene(GAPDH)泳道M：2000bpmarker;泳道1：正常组干细胞；泳道2：病人组干细胞；泳道3：内参基因
12FISHFISHanalysis100×magnification原位荧光杂交图100xA:GreenfluorescencehybridizationsignalinnucleiofCD34+BMHSCsinthepatientgroup,A:病人造血干细胞的细胞核内可见绿色英光杂交信号B:NucleiofHepG22.1.5cellsshowinggreenflourescencsignaB:22.15细胞核内也可见到绿色荧光杂交信号C:AbsenceofgreenfluorescencehybridizationsignalinthenucleiofCD34+BMHSCsinthehealthycontrolsC:正常对照组无荧光信号D:chromosomesofCD34+BMHSCsinpatientgroupD:病人组干细胞染色体E::chromosomesofCD34+BMHSCsinHepG2.2.15cellE:HepG2.2.15cell的染色体F::chromosomesofCD34+BMHSCsincontrolgroupF:健康对照组的染色体
13PhenotypicanalysisofT-lymphocytes(T细胞表型分析）ThefrequenciesofCD3+TCRaβ+weresignificantlylowerinthepatientgroup(31.63±1.65%)thaninthehealthycontrols(41.83±2.85%)onday14(**P<0.01;)whilethefrequencieinthepatientgroup(57.98±1.95%)wasfurtherlowerthaninthehealthycontrol(72.35±1.70%)onday25(***P<0.001;Fig3A).
14CD4+CD8+Tcellsrespectivelyweresignificantlylowerinthepatientgroup(1.28±0.04%)thaninthehealthycontrols(3.44±0.27%)onday14(*P<0.05),Thefrequenciesinthepatientgroup(5.66±1.14%)wasalsolowerthaninhealthycontrols(10.26±1.52%)(**P<0.01;Fig3B)onday25
15CD3+CD4+Tcellsrespectivelyweresignificantlylowerinthepatientgroup(10.27±1.37%)thaninthehealthycontrols(23.51±2.60%)onday14thefrequenciesinthepatientgroup(22.67±4.03%)alsowaslowerthaninthehealthycontrol(44.98±5.96%)onday25(**P<0.01;Fig3C
16However,thefrequenciesoftheCD3+CD8+subsetsweresimilarinthepatientgroup(9.59±2.40%)andinthehealthycontrols(12.30±2.05%)(#P>0.05;Fig3D)onday14and25
17第14天及25天两组由骨髓干细胞诱生的CD4+/CD8+T细胞的比值统计图analysisofCD4+/CD8+TcellgeneratedfromCD34+BMHSCsculturedonOP9-DL1cellsonDay14andDay25
18IncontrastthefrequenciesofCD4+CD25+Tcellswashigherinthepatientgroup(5.15±1.22%)thaninthehealthycontrol(1.82±0.39%)(*p<0.05)thefrequenciesinthepatientgroup(20.00±3.49%)increasedmoreobviouslythaninthecontrols(5.40±0.74%)(**P<0.01;Fig3E).
19CDIandcytokineproduceofT-lymphocytes(增殖指数和细胞子)Patient病人组Control对照组NonPHA(无PHA)AddPHA(加PHA）Non-PHA(无PHA）AddPHA(加PHA)
20TheCDIvaluesislowerinthepatientgroup(1.28±0.06)thanthoseinthehealthycontrolgroup(2.18±0.24)asshownintheFigureA(*P<0.05)TheproductionofIL-2(392.92±122.17pg)andIFN-γ(769.02±99.59pg)waslowerinthepatientgroupthaninthecontrolgroups,asshowninthisFigureB(*P<0.05)
21DISCUSSIONSTheresultsoftheHBSgeneexpressionsuggestedthattheHBVDNAisabletoreplicateintheBMCD34+HSCsofpatientsHBS基因的表达证明乙型肝炎病毒可以感染骨髓造血干细胞并复制FISHanalysissuggestedthatHBSgenesegmentsareirregularlyintegratedintotheapartofHSCsgenomeofthepatients荧光原位杂交结果也证明了乙肝病毒感染了骨髓造血干细胞并且和干细胞的染色体整合Theresultrevealeddown-regulatedofCD3+TCRaβ+,CD3+CD4+,CD4+CD8+andup-regulatedCD4+CD25+TregsbothintheearlyandlatestageofdevelopmentinthepatientgroupT细胞发育的早期和晚期均发生了CD3+TCRaβ+,CD3+CD4+,CD4+CD8+表达下调而CD4+CD25+Tregs表达上调
22ThisobservationsupportsthatabnormalexpressionofTcellphenotypecouldbepresentinbothmatureandimmaturestageofdevelopmentandthatdifferentiationanddevelopmentoftheseTcellsubsetsbecamemoreandmoredisproportionalovertimedevelopmentofTcells.实验结果认为:T细胞在发育时期发生的表型表达异常随着T细胞的成熟表现得越来越明显ManypreviousinvestigatorsindicatedthatchronicHBVinfectionpatientshavealsobeenreducednumbersofCD3+andCD4+cells,CD4+/CD8+ratiosandhighernumbersofcirculatingCD4+CD25+Tregsinperipheralblood,butinourstudythedisproportionofTcellsubpopulationwerealreadypresentduringtheTcelldevelopment已有大量文献报道慢乙肝病人外周血T细胞的CD3+,CD4+，CD4+/CD8+表达下降，CD4+CD25+Tregs表达增高,但是按着我们的研究这种表型异常在T细胞发育期间就已经出现了
23ThelowerIFN-γandIL-2productionandreducedproliferationinthepatientgroupindicatedthatboththeTcellfunctionwerealsodefectiveinthepatientgroup检测结果发现这种诱导后的T细胞分泌的IFN-γ和IL-2也明显减少，说明T细胞的功能也是不足的。WhyisthatthephonotypeandfunctionoftheTcellsofpatientgroupbecomeabnormal?Thereweretwopossiblereason我们分析这种诱导的T细胞缺陷的原因主要有两个
241.continualexpressionofHBsAgbecauseofreplicationandintegrationofHBVDNAinHSCsasaexogenousantigenicexcitationcouldaffectpositiveandnegativeselectionduringTcelldevelopment,whichcouldleadtoabnormalexpressionofTcellphenotypeorapoptosisofsomeTcellssubpopulation由于病毒在干细胞内复制同时表达HBsAg，这种外来抗原在T细胞发育阶段会影T细胞的阳性和阴性选择从而使一些亚群发生程序性的凋亡，因此出现表型表达紊乱2.thesomeHBVDNAgenesegmentsasexogenoussequencesareirregularlyintegratedintotheHSCsgenome,whichcouldleadtogenerearrangementofHSCs.Therefore,theHSCswithchromosomalvariationcouldbecausetodifferentiateintoabnormallymphaticprogenitorcellsandthisprocesscouldinteractthegenerearrangementofTCRaβTcells乙肝病毒的基因片段作为一种外来基因整合到干细胞染色体上，造成这个干细胞发生基因重排，那么这种基因突变的干细胞分化成为病态的淋巴祖T细胞，进而在淋巴祖T细胞表达TCR时发生基因重排也将受到影响从而造成TCR的表达异常
25Accordingly,wepresumedthatTcelldefectsassociatedwithinfectionandintegrationofHBVDNAinHSCscouldbeimprovedbyhematopoieticstemcelltransplantationledtoimmunologicreconstitution因此我们推断慢乙肝病人的T细胞缺陷可能通过造血干细胞移植而改善However,accordingtoourresults,immunereconstructionisunlikelyafterautologoushematopoieticstemcelltransplantation(AHST)inCHBpatient,becausethoseHSCscouldgeneratedefectiveTcell.ThereforeallogeneichematopoieticstemcelltransplantationcouldbemoresuitablethanAHSCT从我们的实验结果来看自体干细胞移植是达不到免疫重建的目的，因为这种干细胞会诱生缺陷的T细胞，因此异基因移植可能是更适合的ItisnotcleariftheeliminationofHBVDNAintheBMHSCscanrestorenormaldifferentiationofHSCsinpatients,Therefore,moreresearchisneededinthefuture如果能清除病人干细胞内的病毒后是否能恢复病人干细胞的免疫重建功能还不清楚，还需要在后续继续研究和探讨
26CONCLUSIONwehaveshownthattheHSCsofchronicHBVinfectionpatientsinfectedandintegratedwithHBVDNAgenerateddefectiveTcellsbecauseofaffectionondifferentiationanddevelopmentofT-lymphocyte.我们认为:乙型肝炎病毒可以感染骨髓造血干细胞而且可以复制和干细胞染色体整合，这种情况由于干扰了造血干细胞的分化而导致骨髓造血干细胞诱生了缺陷的T细胞ThisstudyhasprovidedatheoreticalbasisfortheuseofhematopoieticstemcelltransplantationasatherapyforchronicHBVinfectionpatients我们的研究为未来用造血干细胞移植的方法治疗慢乙肝提供了理论依据
27Thankyou!
