1 3 5 6 6實驗結(jié)果(附論文圖片) 26 371促紅細胞生成素對糖尿病兔心肌間質(zhì)重塑的影響40只健康日本大耳兔禁食12小時，經(jīng)耳緣靜脈采用兩次法注射四氧嘧啶(ALX),按50mg/kg,間隔1周后再按100mg/kg注射ALX,注射后即刻50%葡(1)假手術(shù)組A(2)手術(shù)對照組B(3)手術(shù)用藥組C注射稀釋濃度1000U/ml),3天后再次注射2糖尿病模型成功33只，觀察期間死亡3只，30只進入心梗模型建立階段，心免疫組織化學染色Timp-1積分光密度值(IOD)B組與C組無明顯差別3indiabetesmellitusrabbitsaftertheinductionoligationoftheleftdescendingcoronaryartery.Toexplorethecardioprotective40healthJapaneserabbitswereathefastingplasmaglucoseundertheglucoseoxidthreegroups:shamopegroupC(10).MyocardialinfractionmodelwasestablishedinmodelrabbitsweresubjectedtoligationorabbitsinthegroupCreceicollagenvolumefractionweretested.Specimdeterminationofmatrixmetalloproteinase-9andTissueinhibitoTheaveragechangesinhematocritindiabetesmellitusrabbitstrea4EPO-treatedrabbitsondays14werestatisticallysingnigroupcomparedwiththoseinoperationcontrolgroup,thedecreasionmosInfarctsizecalculatedasfractionEPO-treatedgroupthanoperationgroup(p<0.05).(4)Fourweeksaftermyocardialinfraction,theproteinexprweresignificantlyhigherthanthatincontrolgroup(p<0.05),andtheprexpressionsofMMP-9ingroupCweproteinexpressionsofTimp-1ingroupcIncreasedexpressionsofMMP-9,interstitialfibrosisanddegradedleftventricularfunctionmayplayanimportantroleincardiacremodelingfandmyocardialinfarction.Earlyinandcollagenvolume,preventErythropoietin;diabetesmellitus;myocardialinfarction;extracellularremodeling;matrixmetalloproteinase-9;Tissue-InhibitorofMetalloproteinase-15中文全稱Matrixmetalloproteinas組織型蛋白酶抑制劑-1四氧嘧啶急性心肌梗死糖尿病6促紅素對糖尿病兔心梗后心肌間質(zhì)重塑的影響實驗材料與方法一、實驗材料7(二)主要器械及儀器(3)電子天平、恒溫箱、冰箱(5)強生穩(wěn)豪血糖儀及試紙(6)圖像分析系統(tǒng)(AdvancedImageSystem)(4)二抗、DAB染色劑(5)速眠新注射液(6)PBS液、10%甲醛(7)普通肝素注射液(8)碘伏消毒液、生理鹽水(9)促紅細胞生成素(10)四氧嘧啶(11)乙醇、戊巴比妥(12)二甲苯、過氧化氫(13)10%水合二醛(15)0.2%冰醋酸水溶液8(16)1%磷鉬酸水溶液(17)1%光綠水溶液二、實驗方法(一)實驗分組(1)假手術(shù)組A(2)手術(shù)對照組B(3)手術(shù)用藥組C(二)動物模型的建立方法1、糖尿病模型四氧嘧啶(ALX),按50mgkg,間隔1周后再按100mgkg經(jīng)耳緣靜脈注射ALX。2、心肌梗塞模型的建立9射器抽凈胸腔內(nèi)殘留氣體，保證術(shù)后肺通氣功能。術(shù)后連續(xù)3天肌肉注射青霉素80萬單位抗感染，附手術(shù)圖片(圖1)。暴露心臟，迅速切下心臟，用生理鹽水(4℃)沖洗，找到結(jié)扎線，沿左室長軸垂直方向?qū)⒆笮氖覚M行切成2份，靠心尖部標本放入10%甲醛溶液固定24-48小時，三、檢測指標(一)檢測血紅蛋白濃度(2)手術(shù)后用藥14天檢測血紅蛋白濃度面至心底橫向切成薄片每個標本4片(厚度約0.8～1.0mm)。將心臟薄片浸入36-37℃1%TTC(氯化三苯基四氮唑)磷酸鹽緩沖液中孵化30min。存活組織中含(三)常規(guī)蘇木素伊紅染色(HE染色)·(1)脫蠟及脫苯：將干燥的切片放入二甲苯I10-20min,II20min脫蠟。用100%無水酒精I脫苯10min,然后放人100%無水酒精Ⅱ脫苯(3)0.01M的PBS洗1遍2分鐘。(8)0.01M的PBC洗3次每次3分鐘。(9)滴加試劑SABC,37℃溫箱30分鐘。(10)0.01M的PBS洗3次每次4分鐘。(12)蒸餾水洗2遍。(13)蘇木素復染2分鐘(14)蒸餾水洗2遍。(3)用Regaud蘇木精染液染核5-10min(7)1%磷鉬酸水溶液分化3-5min(8)苯胺藍染色5min40只，成功30只，死亡6只，考慮為高血糖酮癥酸中毒或低血糖所致，4只未達組死亡4只，C組死亡2只。最終各組存留動物分別為7只、7只、8只。(2)用藥后血紅蛋白濃度測定表1,手術(shù)后用藥14天檢測血紅蛋白濃度(*±s)gL組別n血紅蛋白899對照組(B組)心肌梗死范圍相比有統(tǒng)計學意義(p<0.05)(見表2,圖2A-C)。組別N心肌總面積百分比7078注：手術(shù)用藥組與手術(shù)對照組比較*p<0.05較B組較少HE染色(×200)心肌細胞間少量藍染纖維素染色(×400)塊狀SABC法(×400)法(×400)法(×400)法(×400)表3,圖像分析及統(tǒng)計學處理結(jié)果組別N778注：手術(shù)用藥組與手術(shù)對照組及假手術(shù)組有顯著差異*p<0.05手術(shù)用藥組與手術(shù)對照組無顯著差異#p>0.05。正常情況下，心肌間質(zhì)成纖維細胞(fibroblast)負責膠原的合成。然而，研究發(fā)基質(zhì)產(chǎn)生細胞。其功能及數(shù)量的改變也與多種器官(皮膚，肝臟，肺臟，腎臟)的ECM沉積和纖維化有關(guān)[123]。Myofibroblast是活化的成纖維細胞，兼具有成纖維細胞和平滑肌細胞特性，在糖尿病心臟及心肌梗心肌中均有myofibroblas存在，myofibroblas在修復的組織中大量長生膠原4-9,它的形成受多種生長因子、耗氧量、擴張冠狀動脈、抗凝、溶栓等防止病變的進展，應用經(jīng)皮腔內(nèi)冠脈成形術(shù)及支架植入和冠脈搭橋術(shù)恢復局部冠脈血流。發(fā)生急性心肌梗死后，即便是采國外研究報道血管緊張素轉(zhuǎn)換酶抑制劑(ACE長，其生血管的作用和內(nèi)皮生長因子相似，并且其研究發(fā)現(xiàn)EPO新生血管的作用無法在心梗術(shù)后24小時內(nèi)發(fā)揮作用，即新生血管的作用不可能引起早期24小可以引起血管擴張和促進側(cè)枝循環(huán)這可以在心梗早期減少心肌梗死面積。因此惡化心肌收縮功能，通過本實驗masson染色發(fā)現(xiàn)EPO干預組心等1的研究中發(fā)現(xiàn)，心梗后慢性心衰大鼠模型中心肌中炎癥因子水平升高，并且多都參與了心肌重構(gòu)。炎癥細胞因子可以誘導氧化應激，如TNF-2可以直接誘因子的表達，因此在心衰的發(fā)展中形成了惡性循450。Li等研究發(fā)現(xiàn)在心衰大氧化的DNA損傷標志物8-OhdG,減少炎癥因子I-1β,IL-6,TNF-a干預組EPOR的下游靶信號Stat3,Stat5,Akt顯著激活，在體癥細胞因子的產(chǎn)生，可以推測EPO通過Jak/Stat信號通路抗炎癥反應，通過區(qū)由于進行性ECM合成增加，尤其是I型膠原大量沉積和I/Ⅲ型膠原比例升高心肌ECM的損害和喪失是MI后心室重構(gòu)的一個決定因素。而存在心肌中的ECM,引起ECM退化，進而導致心肌細胞排列紊亂，從而引起心室的重構(gòu)。程度明顯減輕。本試驗中通過免疫組化觀察蛋白表達研究發(fā)現(xiàn)，EPO干預組用。1DesmouliereA,DartbyIA,GabbianiGNo2MitchellJ,Woodcock-MitchellJ,ReynoldsS,etal.Alph-smoothmparenchymalcellsofbleomycininjuredratlung[J].LabInv3HewitsonTD;WuHL,BeckerGJ,etal.Interstitialmyofibroblastinfectionandscarring[J].AmJNephrol,1995,15(5):411-417.4CalderoneA,BellhandS,DrapeauJ,etal.Scarmyofibrobexpressnatriureticpeptides[J].CellPhysiol,2006,207:5BadidC,MounierN,CostaAM,etal.Roleofmyfibroblexcessivescarring:interestoftheirassessmentinnephropathies[J].HistolHistopathy,2000,6TomasekJI,GabbianiG,HinzB,etal.Myofibroblastsandtissueremodeling[J].NatRevMolBiol,2002,3(5):349-37LunenR,PetroV,etal.TransforminggrowthfactocultresofcardiacfibroblastsistheresultoftheappearanceofmyofibroblastFindExpClinPharmacol,2002,24(6):339YoussoufianH,LongmoreG,NeumannD,etal.Structure,function,andactivationoerythropoietinreceptor[J].Blood,1993,81:2223-22310WuWC,RathoreSS,WangY,etal.Blomyocardialinfarction[J].NewEnglandJMed,200111CheungWK,GoonBL,GuilfoileMC,etal.Pharmacokineticsandpharmacodynamicsrecombinanthumanerythropoietinsubjects[J].ClinPharmacolTher,1998,64:412-423.12ItsikBD,BrittaH,ShmuelF,etal.RepeatedLow-doseofErythropoietinisAssociaImprovedLeftVentricularFuncmRNAexpressionrelatedtocardiacremod14YoshiyamaM,TakeuchiK,OmuraT,etal.Effec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升高10%。接受血2MarontAMBiochemicalcharacteristics,biologicaleffects,indictionsandresultsofushematology[J].Tumor.1997Jul-Aug;83:3-15.3汪東海等.重組人促紅細胞生成素中唾液酸含量對體內(nèi)生物活性的影響[J].中國生物制品4KaltwasserJP,KesslerU,CottsohalkR,elal.Effectofrecombinanthumane2001,28:2430-2436.7RibattiD,PrestaM,VaccaA,etal.H8JaquetK,KrauseK,Tawakol-Khoangiogcnicpotential[J].MicrovascularRes,2002,64(2):326-3339RabattiD,MarzulloA,NicoB,etal.Ercarcinoma[J].Histopathology,2003,42(2):246-25010StohlawetzPJ,DzirioL,Hergovithrombopiesisinhumans[J].thevascularendothelialfunctioninanaesthetizedrabbits[J].BrJPharm12BarrettJD,ZhangZ,ZhuJH,etal.Erythropoietinregulateapoptosisbyaphosphatidylinositol3kinase-dependentpathway14WaldM,GutniskyA,BordaE,etal.Eryt