促紅細(xì)胞生成素對(duì)糖尿病兔心梗后心肌間質(zhì)重塑影響

1 3 5 6 6實(shí)驗(yàn)結(jié)果(附論文圖片) 26 371促紅細(xì)胞生成素對(duì)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(shí)，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(duì)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(jìn)入心梗模型建立階段，心免疫組織化學(xué)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(duì)糖尿病兔心梗后心肌間質(zhì)重塑的影響實(shí)驗(yàn)材料與方法一、實(shí)驗(yàn)材料7(二)主要器械及儀器(3)電子天平、恒溫箱、冰箱(5)強(qiáng)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(xì)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(shí)驗(yàn)方法(一)實(shí)驗(yàn)分組(1)假手術(shù)組A(2)手術(shù)對(duì)照組B(3)手術(shù)用藥組C(二)動(dòng)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(zhǎng)軸垂直方向?qū)⒆笮氖覚M行切成2份，靠心尖部標(biāo)本放入10%甲醛溶液固定24-48小時(shí)，三、檢測(cè)指標(biāo)(一)檢測(cè)血紅蛋白濃度(2)手術(shù)后用藥14天檢測(cè)血紅蛋白濃度面至心底橫向切成薄片每個(gè)標(biāo)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(xiàn)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(fù)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(lán)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(dá)組死亡4只，C組死亡2只。最終各組存留動(dòng)物分別為7只、7只、8只。(2)用藥后血紅蛋白濃度測(cè)定表1,手術(shù)后用藥14天檢測(cè)血紅蛋白濃度(*±s)gL組別n血紅蛋白899對(duì)照組(B組)心肌梗死范圍相比有統(tǒng)計(jì)學(xué)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(duì)照組比較*p<0.05較B組較少HE染色(×200)心肌細(xì)胞間少量藍(lán)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(jì)學(xué)處理結(jié)果組別N778注：手術(shù)用藥組與手術(shù)對(duì)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(duì)照組無顯著差異#p>0.05。正常情況下，心肌間質(zhì)成纖維細(xì)胞(fibroblast)負(fù)責(zé)膠原的合成。然而，研究發(fā)基質(zhì)產(chǎn)生細(xì)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(xì)胞，兼具有成纖維細(xì)胞和平滑肌細(xì)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(fù)的組織中大量長(zhǎng)生膠原4-9,它的形成受多種生長(zhǎng)因子、耗氧量、擴(kuò)張冠狀動(dòng)脈、抗凝、溶栓等防止病變的進(jìn)展，應(yīng)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(fù)局部冠脈血流。發(fā)生急性心肌梗死后，即便是采國(guó)外研究報(bào)道血管緊張素轉(zhuǎn)換酶抑制劑(ACE長(zhǎng)，其生血管的作用和內(nèi)皮生長(zhǎng)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(shí)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(kuò)張和促進(jìn)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(shí)驗(yàn)masson染色發(fā)現(xiàn)EPO干預(yù)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(xì)胞因子可以誘導(dǎo)氧化應(yīng)激，如TNF-2可以直接誘因子的表達(dá)，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(biāo)志物8-OhdG,減少炎癥因子I-1β,IL-6,TNF-a干預(yù)組EPOR的下游靶信號(hào)Stat3,Stat5,Akt顯著激活，在體癥細(xì)胞因子的產(chǎn)生，可以推測(cè)EPO通過Jak/Stat信號(hào)通路抗炎癥反應(yīng)，通過區(qū)由于進(jìn)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(gè)決定因素。而存在心肌中的ECM,引起ECM退化，進(jìn)而導(dǎo)致心肌細(xì)胞排列紊亂，從而引起心室的重構(gòu)。程度明顯減輕。本試驗(yàn)中通過免疫組化觀察蛋白表達(dá)研究發(fā)現(xiàn)，EPO干預(yù)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,andactivationoerythr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.3區(qū)，由8個(gè)外顯子構(gòu)成，長(zhǎng)約6kbp。cDNA序列分析表個(gè)氨基酸的信號(hào)肽、266個(gè)氨基酸的末端部分、22個(gè)氨基酸的跨膜部分和263個(gè)氨基酸的胞漿區(qū)組成。經(jīng)轉(zhuǎn)染細(xì)胞系(COS細(xì)胞系)cDNA表達(dá)可產(chǎn)生66KDa508可能是由于后者糖基化所引起。EPO受體含有由4個(gè)保守性半胱氨酸殘基組成β亞單位等人EPO受體cDNA及蛋白質(zhì)一級(jí)結(jié)構(gòu)序列與鼠之間存在82%的同源stimulatingprotein,NESP),其受體仍為EPOR,優(yōu)點(diǎn)是血漿半衰期較EPO延長(zhǎng)大約放射性碘標(biāo)記的RHEPO研究證明每個(gè)血管內(nèi)皮細(xì)胞大約有27000個(gè)EPOR。生MMP-2,促進(jìn)其增生，激活細(xì)胞JAK2,促進(jìn)該細(xì)胞分化形成血管結(jié)構(gòu)等。肌毛細(xì)血管的生長(zhǎng)。應(yīng)用濃度為2.5U/ml的EPO可使毛細(xì)血管的生長(zhǎng)增加220%,表達(dá)上調(diào)，使VSMC對(duì)血管緊張素Ⅱ的敏感(三)EPO與心肌氨酸激酶、蛋白激酶C及磷脂酶C抑制劑作用后(四)抗心肌細(xì)胞凋亡(五)對(duì)心肌梗死后心室重構(gòu)及心功能的影響(六)其它作用(七)EPO在心血管系統(tǒng)中的不良反應(yīng)者在開始EPO治療后，血壓可能升高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(xì)胞生成素中唾液酸含量對(duì)體內(nèi)生物活性的影響[J].中國(guó)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