急性创伤性深静脉血栓消退与不消退差异表达基因研究

急性创伤性深静脉血栓消退与不消退差异表达基因研究中文摘要目的：在建立大鼠急性创伤性肢体深静脉血栓动物模型基础上，应用Affymetrix基因芯片技术，从基因水平研究血栓形成后，消退与不消退两种不同病理过程间股静脉中差异表达基因，探索影响创伤性肢体深静脉血栓消退的部分关键因素。方法：1.分组：250只SD大鼠（雌雄不限）随机取20只作为对照组，余230只据创伤造模后血栓形成病理变化过程分为：创伤即刻组（B组，造模后0.5小时）、血栓形成前期组（C组，造模后2.5小时）、血栓形成高峰期组（D组，造模后25小时）、血栓消退期组（E组，造模后72小时）、血栓不消退组（F组，造模后72小时）和血栓不形成组（G组，造模后72小时）。2.造模：对照组大鼠不造模，创伤组大鼠造模时不行麻醉，造模方法为：双侧腹股沟区碘伏液消毒后行内侧切口，长约1cm，暴露出股动、静脉及股神经，稍作钝性分离显露长约1.5cm的股静脉，用12.5mm全齿蚊式血管钳分三段各钳夹血管1次（力量为血管钳紧1扣，每次持续3秒）后，用1号丝线全层间断缝合皮肤切口，不放置引流，除对照组、创伤即刻组外大鼠均行双后肢髋人字石膏固定；造模后观察大鼠双足颜色和肿胀情况。3.取材方法：对照组大鼠不造模即取材，创伤即刻组于造模后0.5小时，其余各组大鼠在相应时相点沿原切口暴露股静脉，观察血栓形成状态，符合分组标准后，用3％的戊巴比妥钠溶液按1ml/kg体重，腹腔内注射麻醉，仰卧位固定，碘伏液消毒双后肢前内侧皮肤区域后，沿双侧股静脉走行切开皮肤约4.5cm，观察局部组织反应、股静脉血栓是否形成、严重程度及消退、不消退情况，分别记录；显微镜下分离并切取双侧各长约4.5cm的股静脉及主要属支，留长约0.5cm的血管用甲醛固定，送HE染色组织切片镜检；0.9%生理盐水冲洗干净血管中的血液或血栓，离体30秒内放入冻存管，置入液氮罐中保存，用于总RNA提取。4.总RNA提取：TRIzol法分别提取上述7组股静脉标本总RNA；各组总RNA样品经琼脂糖凝胶电泳检测合格后（28SRNA和18SRNA条带整齐，两者带宽比值超过2倍）分为两份，一份送以进行基因芯片检测，另一份备用以行RT-PCR检测。5.芯片及RT-PCR检测：按AffymetrixRAT230A表达谱芯片操作流程，经cDNA探针制备、杂交、洗脱、染色、扫描，完成芯片检测；应用RT-PCR技术检测保留的RNA样品中IL-1β、Cinc2表达情况，并与芯片数据作比较。6.数据筛选及分析：通过倍数变化分析方法（两组间同一基因的Signallog2ratio比较，差值必须≥1或≤-1，常规认为具有差异性），选取血栓消退与不消退组差异表达基因，应用GeneCluster3.0分析软件聚类（能更容易地选取共表达的基因组，提示可能具有相似功能），绘制直观曲线图，并行GO功能分类，查询“NCBI”、“CNKI”网站及期刊文献，搜寻相应背景知识资料，结合表达变化规律综合分析。结果:1.250只SD大鼠共死亡5只，其中3只因造模过程中股动脉破裂，出血死亡，造模后25小时内2只死于肺栓塞，其余大鼠均存活；D、E、F、G四组190只大鼠死亡5只，25小时有126只血栓形成，D组取材用去25只，余101只观察至72小时，64只血栓消退，37只血栓不消退；至72小时又有23只发生血栓，36％，血栓不形成率19.46％；HE染色股静脉组织切片镜检证实：大体观察血栓形成状态进行的分组准确可靠。2.各时相点7份标本总RNA经琼脂糖凝胶电泳检测，28SRNA和18SRNA条带整齐，两者量的比值超过2倍，样品质量好、无降解。3.AffymetrixRAT230A芯片所能检测的15866个基因中，7389个基因有差异表达，占总数的46.57%，全部时相点均无变化的有8477个，占总数的53.43%；主要涉及促分裂素原活化蛋白激酶、Ca++、黏附斑、刺激神经的配体-受体相互作用、细胞因子-受体相互作用、核糖体、肌动蛋白细胞骨架、Wnt、嘌呤代谢、白细胞经内皮迁移、胰岛素、糖酵解/糖异生等信号通路。TostudythedifferentialexpressiongenesbetweenthrombiresolutionandinsolutionintheprocessofacutetraumaticdeepveinthrombosisbygenechipAbstractObjectives:Basedonestablishingaratmodelofacutetraumaticlimbdeepveinthrombosis.ThroughtheAffymetrix230Agenechip,tostudythedifferentialexpressedgenesbetweenthethrombiresolutiongroupandthrombiinsolutiongroupafterthrombosis.Toexplorethepartialinfluencefactorsofresolutionintraumaticdeepveinthrombosisonthelevelofgene.Methods:1.Grouping.20SDratsfromthetotal250(non-restrictionfemaleandmale)weredividedrandomlyintothecontrolgroup;theremained230weredividedinto6groups:traumainstantgroup(B,at0.5haftermodeling),thrombosisprophasegroup(C,at2.5haftermodeling)，thrombosiscrest-timegroup(D,at25haftermodeling),thrombiresolutiongroup(E,at72haftermodeling),thrombiinsolutiongroup(F,at72haftermodeling),non-thrombosisgroup(G,at72haftermodeling)accordingtodifferentphasesaftermodelbeingproducedandtheresultsofpreliminaryexperiment.2.Modeling.Ratswerenotanesthetizedinmodelproducingprocess.AfteringuinalregionssterilizedbyIodophors,1cmlongmedialinguinalgrooveincisionwasadoptedtoexposeproximalfemoralvein,arteryandnerve.Afterbluntdissection,1.5cmexposedfemoralveinwasclampedatthreepointsseparatelywith12.5mmmosquito-hemostaticforceps,onceeachpoint;theclampingstrengthwasfasteningonebarbofhemostaticforceps,lasting3secondseachtime,thentheincisionwassutured,nodrainagewasset.RathibateralposteriorlimbswerefixedwithhipspicacastsexceptforgroupAandB.Atdifferentphasesaftermodelbeingproduced,colorandswellingextentofbothfeetwereobservedbygrossobservation.3.Methodsforobtainingfemoralveins.Ratswereanesthetizedwith3%pentobarbitalsodium(1ml/kg,intraperitonealinjection),supinepositionfixation,sterilizinganteromedialskinofhibateralposteriorlimbsthroughiodophors,exposinghibateralfemoralveins.IngroupA,4.5cmfemoralveinandrelatedmaintributarieswereresected.IngroupB,at0.5h;groupC,at2.5h;groupD,at25h;groupE,F,G,at72haftermodelbeingproduced,thesameregionvasculartissuewasalsoresectedseparately.Partofthe0.5cmvesselseparatedforHEstainingpathologicalhistologicalanalysis,therestwererinsedby0.9%physiologicalsalinetocleanupbloodandthrombi.Thevesselspecimenswereputintonitrogencanisterinlessthan30secondsafterexvivo,whichwouldbeusedfortotalRNAextraction.4.ExtractionoftotalRNA.Afteraffirmingthestatusofthrombosisbyhistologicalanalysis,totalmRNAsoffemoralveinspecimensfromthe7phaseswereextractedseparatelythroughTRIzolmethod.AlltotalRNAsampleswerecheckedbyagarosegelelectrophoresisanddevidedintotwoportionsforbeingdetectedthroughgenechipandRT-PCR.5.RNAdetectionthroughgenechipandRT-PCR.ThroughcRNAprobespreparation,hybridization,washing,stainingandscanningwereperformedorderlytofinisharraydetectingaccordingtotheoperationflowsheet.TovalidatetheaccuratissimeofgenechipthroughRT-PCR.6.Datascreeningandanalysis.Afterperformingtherestrictiveconditions:=1\*GB3①thedataofexperimentalgroupinEvsAandFvsAshouldnotbemarked“absent”(thatistosay,thesignalintensityisweak);=2\*GB3②Signallog2ratiosofthesamegenecomparedbetweentheresolutiongroupandinsolutiongroupmustbe≥1or≤-1(representingvariabilityroutinely),tosearchoutthedifferentialexpressiongenesbetweentheresolutiongroupandinsolutiongroup.ThescreenedgeneswereclusteredthroughsoftwareofGeneCluster3.0anddrawnvisualpicture.Thesegenesfunctionswereinquestedfromweb“NCBI”,“CNKI”,etc.andaggregateanalysiswasdone.Results:1.3and2ratsdiedbecauseoffemoralarteryruptureandpulmonaryembolismrespectively.IngroupA,B,C,noratpresentedthrombosis.Inthe190ratsofgroupD,E,FandG,total5ratsdied.From2.5hto72haftermodelbeingbuilt,149ratspresentedthrombosis,36werenothrombosis,64presentedthrombiresolutionand37presentedthrombiinsolution.Themortalityofratsis2％,incidenceratesofthrombosis,non-thrombosisandresolutionare80.54％,19.46%and63.37％respectively.2.ThetotalRNAsamplesof7groupswereprovedtobehighqualitieswithoutdegradation.occurrence3.Thehybridizationsignalintensityofarrayswassatisfactory.ThechangetendencyofIL-1βandCinc2detectedbygenechipandRT-PCRwereingoodcoincidence.4.Inthe15866ratgeneswhichcanbedetectedbyAffymetrixRAT230Amicroarray,7389presenteddifferentialexpression,wereinvolvedinMAPK,Calcium,Focaladhesion,etc.signalingpathways;8477didnotpresentdifferentialexpressioninallphases.5.Aftercomparisonbetweengroupresolutionandgroupcontrol,24genesupregulated(Log2Ratio≥4.0).Amongthem,14geneshavenofunctionaldescription,10genes(Top2a、Stk6、Slpi、Olr1、Kdap、Itgam、Il6、Cd8a、Brca1、A2m)withpartialGOfunctionaldescriptionrefertoDNAmetabolism,proteinmetabolism,inflammatoryreaction,etc..9genesdownregulated(Log2Ratio≤-4.0).Amongthem,4geneshavenofunctionaldescription,5genes(Lepr、Gpd3、Atp1b4、Af6、Adh7)withpartialGOfunctionaldescriptionrefertocell-cellsignaling,energymetabolism,etc..6.Aftercomparisonbetweengroupinsolutionandgroupcontrol,26genesupregulated(Log2Ratio≥4.0).Amongthem,16geneshavenofunctionaldescription,10genes(Slpi、Olr1、Nos2、Mmp9、Kdap、Itgam、Il6、Cxcl2、Cinc2、Cd8a)withpartialGOfunctionaldescriptionrefertoDNAinflammatoryreaction,cell-cellactionetc.;8genesdownregulated(Log2Ratio≤-4.0).Amongthem,6geneshavenofunctionaldescription,2genes(Gpd3,Cyp1a1)havepartialGOfunctionaldescription.7.The118differentialexpressiongenesbetweentheresolutionandinsolutionhadbeensearchedout.Amongthem,48genes,whichwithsymbolswereassignedGOfunctionsbyGenebankandwererelatedtothefunctionsofapoptosis,tumor,binding,metabolism,cellcycling,signaling,constructionmolecularactivityandtransporter.8.Comparedtheresolutiontoinsolution,theexpressionsofMmp-9,Mmp-12,Mmp-13,IL-1,Cxcl2,Cinc2,Ccl3,PtgesandArginasewereconspicuouswhichexpressedhigheringroupsofDandFandloweringroupsEandGinversely.Conclusions:1.ThefemoralveinexpressMmp-9,Mmp-12,Mmp-13relatedtotissuerepair,angiopoiesis,andIL-1,Cxcl2,Cinc2,Ccl3relatedtoinflammationtopromotevascularrepairandenhanceanticoagulation.ItexpressPtges,Arginaserelatedtovasodilatationandinhibitionofplateletaggregationtoregulateincommonthethrombiresolutionthroughchangingthestatusoflocalvessel.Whethertheprocessofthrombiresolutionispromotedorpreventedbythemshouldbeprovedthroughmorefunctionalexperimentsatgeneor/andproteinlevel.2.Thethreeunknownfunctiongenes(1391505_x_at、1379497_at、1377365_at)coexpressconspicuouslywiththeabovegenes.Itispresumedthatthefunctionofthemarerelatedtotissuerepair,angiopoiesis,inflammation,vasodilatationandinhibitionofplateletaggregation.3.Theratmodelofacutetraumaticdeepveinthrombosiscanbeestablishedthroughclampingfemoralveinwithhipspicacastfixation.4.TheratacutetraumaticdeepveinthrombosisisacomplicatedprocesswhichinvolvesinMAPK,calciu