治療超聲聯(lián)合微泡溶解體外血栓實(shí)驗(yàn)研究

治療超聲聯(lián)合微泡溶外血栓的實(shí)驗(yàn)研AStudyonEfficacyofTherapeuticUltrasoundandMicrobubbleContrastAgentonAugmentationofThrombolysis：朱莉玲 師：柳建華教 業(yè)：影像醫(yī)學(xué)與核醫(yī)20125月·英文縮略詞表英文縮寫英文全稱中文全稱ExternalTherapeuticUltrasoundContrastDutyHematoxylinHighIntensityFocusedAcuteMyocardialIntracranial顱內(nèi)治療超聲聯(lián)合微泡溶外血栓的實(shí)驗(yàn)研中研究目材料與方法取健康全血共112ml，分成112份，每份約1ml，置于1.5m1塑料EP管6檔(5.47MPa)，將超聲治療儀的占空比0.216%、1.080%、驗(yàn)第二部分：固定超聲治療儀的占空比為7.200%，將超聲治療儀的超聲聲壓分別設(shè)6檔(5.47MPa)、3檔(3.75MPa)、1檔(2.80MPa)，并根據(jù)有無微泡注入共（W1，計(jì)算各組的溶栓P=[(W0—W1)/W0]×100%。明顯統(tǒng)計(jì)學(xué)意義。所有數(shù)據(jù)均采用SPSS13.0統(tǒng)計(jì)軟件進(jìn)行。AStudyonEfficacyofTherapeuticUltrasoundandMicrobubbleContrastAgentonAugmentationofThrombolysisWiththedevelopmentofultrasoniccontrastandpreparativetechniqueofmicrobubbleultrasoundcontrastagent(MUCA),ultrasoniccontrastagentisnotonlyusedonthefieldofclinicaldiagnosis,butalsoreceivedgrowingconcernonthefieldofclinicaltreatment,suchasaugmentationofthrombolysis,genetransfectiontotheinfractedmyocardium,drugdelivery,penetrationofbiologicalbarrierandsoon.Theabilityofultrasound(US)todissolvethrombus,eitheraloneorcombinedwithUScontrastagents,hasbeenstudiedinvitroandinvivobyseveralpioneeringgroupsfromthe1990s.Itisclearthe“optimal”ultrasoundparametersforultrasound-enhancedthrombolysis(UET)havenotyetbeenidentified.ThisexperimentwasdesignedtostudytheeffectivenessofMUCAenhancedsonothrombolysisinvitro,andtoexplorewhetherdutycycleandintensityoftherapeuticultrasoundcaninfluencetheeffectofUETcombinedwithMUCA.MaterialsandPreparationof112mlofwholebloodweredrawnfromhealthyvolunteersanddividedinto112parts,eachof1ml,aliquotedinto1.5mlEPplastictube.Plastictubewasplacedin37°Cconstanttemperatureboxforabout3hoursandtheclotswerestoredina5°Crefrigeratorfor3daystoallowcompleteclotretraction.ThentheywerewashedbysalineandweightedaftertheyweredrybyelectronicscalebeforealltheExperimentprotocolsAllexperimentsdescribedinthisstudywereproceededinthe37°Cconstanttemperaturebox.Thetransducerwasdriveninpulsedandfocusedmode.Thetreatmentmodewasfixedatgrade4andthetreatmenttimewasfixedat30minutes.Thewholeexperimentconsistedoftwoparts.Part1Theintensitywasfixedatgrade6(5.47MPa)andthedutycycleDC)wassetat0.216%1.080%3.600%7.200respectively.Inthisparttherewere10treatmentgroupsaccordingtowhetherexposedtoultrasoundormicrobubblesornot.Part2:TheDCwasfixedat7.200%andtheintensitywassetat5.47MPa3.75MPa、2.80MParespectivelyInthisparttherewere8treatmentgroupsaccordingtothepresenceofMUCAornot.Afterthetreatmentwascompleted,thethrombiweretakenoutimmediay,washedbysalineandthenweightedagainbyelectronicscaleaftertheyweredrytoevaluatetheeffectofthetreatmentasthechangeinweightfromthebaselinevalue.Thespecimenswerestainedbyhematoxylinandeosin,andthestructureofclotswasobservedwithalightmicroscope.TheaveragethrombolysisratiooftheMB+USgroupwassignificantlyhigherthantheMBgroupatthesameconditions(P<0.05,P<0.01).Inpart1,withtheincreasingofdutycycle,thethrombolysisratebecamehigherinboththeUSgroupandtheMB+USgroupwhentheintensityisfixedatgrade6.ThethromblysisratewashighestintheMB+USgroupwhenthedutycyclewassetat7.200%(P<0.01).Inpart2,thethrombolysisratebecamehigherwiththeincreasingofintensityinboththeUSgrouportheMB+USgroupwhenthedutycyclewasfixedat7.200%.ThethromblysisratewashighestintheMB+USgroupwhentheintensitywassetat6HEstainedsectionsshowedoriginalstructureofclotsanddisaggregationoffibrousstructureandthedestructionofredcellsinclotafter30min.Therapeuticultrasoundhasthedefiniteeffectivenessofthrombolysis,andtheeffectivenesscanbesignificantlyaugmentedbyintroductionofMUCA.Higherdutycycleandhigherintensitiesmadehigherthrombolysisrateincertainconditions.：Microbubbles;Externaltherapeuticultrasound;Dutycycles;Intensity;Thrombolysis治療超聲聯(lián)合微泡造影對(duì)體外血栓溶解作用的前探測(cè)顱腦。20世紀(jì)70年代后，多功能B超顯像儀的問世，大大提高了超聲的（A）是一種可以通過肺循環(huán)的含有高濃度微氣泡的混懸液，其聲學(xué)特性與血灌注方面的特異性和靈敏度。使用A檢查具有操作簡(jiǎn)便、耗時(shí)短、無電離輻射、無創(chuàng)聲溶栓、增強(qiáng)強(qiáng)度聚焦超聲（FU）的治療作用等方面發(fā)揮作用。超聲溶栓技術(shù)發(fā)展于20世紀(jì)90[17]。研究表明：超聲空化效應(yīng)的發(fā)生是一種閾過程，空化閾是液體發(fā)生空化（AMI融和體表治療性超聲助融等。靜脈注射大量溶栓藥的副作用主要是引起，因材料與方一、材料與試驗(yàn)器血栓：取健康全血共112ml，分成112份，每份1ml裝于1.5m1“脂氟顯”脂膜微泡，第三第二附屬醫(yī)院超聲科研制，氣體為全頻率831KHz，脈沖寬度100-1000微秒可調(diào)，峰值聲壓4.6Mpa，平均聲強(qiáng)電子天平：方瑞儀器的JA系列電子天平（精度0.001g）其它：5ml注射器、濾紙、1.5mlEP二、實(shí)驗(yàn)方案372cm的吸聲海綿，以減少聲波反射,箱內(nèi)盛適量蒸餾水,。血栓（EP管或注射器）平置于栓垂直照射（1）4（4s4s的交替治療方式30min。6共4種，共分成10組。件不變，將超聲治療儀的聲壓分別設(shè)為6檔、3檔、1檔，共分成8組。盡量裝滿EP塑料管。該組不進(jìn)行超聲輻照。1.5mlEP0.5ml造影劑，再加超聲+微泡組：取兩個(gè)5ml的注射器：注射器A和注射器B。將已稱重的血A1ml2.5ml0.5mlB內(nèi),雙手不斷來回搓注射器，以保證生理AB分別與三通管相接。三通管第三個(gè)開口接7號(hào)頭皮針。三通管三個(gè)開口接好后，先關(guān)閉頭皮針的開口，此時(shí)注射器A與注射器B相通，快速推注注射器B，將注射器B1ml微泡液注入注射器A后開始給予超聲治療10min將注射器A內(nèi)的微泡液經(jīng)頭皮1ml后，再次將注射器B內(nèi)的微泡液1ml至注射器A,以維持注射器A內(nèi)的微泡濃度圖 實(shí)驗(yàn)裝置示意圖：所有實(shí)驗(yàn)過程均在37℃的恒溫箱中進(jìn)行，在恒溫箱底部各組30min后取栓，并用生理鹽水沖洗、濾紙吸干、稱重（W1，計(jì)算各組的溶栓率P=[(W0—W1)/W0]×100%。三、病理學(xué)檢查四、統(tǒng)計(jì)學(xué)處理溶栓率均以均值±標(biāo)準(zhǔn)差（XS）表示，兩組間比較采用配對(duì)t明顯統(tǒng)計(jì)學(xué)意義。所有數(shù)據(jù)均采用SPSS13.0統(tǒng)計(jì)軟件進(jìn)行。 一、溶栓率比較：P>0.05；無超聲無微泡組與各組單純超聲組的溶栓率比較差異有非常顯著性意義，P<0.01；單純超聲組（0.216%、1.080%、3.600%、7.200%）7.200%P7.200%）組間的溶栓率比較，P<0.01，差異有非常顯著性意義。上述結(jié)果說明空比越高的溶栓率越高，占空比設(shè)為7.200%的第6組溶栓率最高%<0.01組組間的溶栓率比較，占空比越高時(shí)溶栓率越高。治療超聲占空比設(shè)為%（P<0.01無微泡組與各組單純超聲組的溶栓率比較，P<0.01,差異有非常顯著性意義；單純超聲組（6檔、3檔、1檔）P<0.01，差異有非常顯著性意義；單純超聲組與超聲+微泡組的溶栓率比較（聲壓分別為6檔、3檔、1檔）P<0.01，差異有非常顯著性意義；超聲+微泡組（6檔、3檔、1檔）組間的溶栓率比較P<0.01，差異有非常顯著性意義。恒定治療超聲（7.200%的溶栓率越高，超聲強(qiáng)度設(shè)為6檔的第5組溶栓率最高（該組溶栓率為%（P<0.01組間的溶栓率比較，超聲強(qiáng)度越大的組溶栓率越高。超聲強(qiáng)度設(shè)為6檔的第%（P0.01表1 溶栓率比較（X±S，n=8）實(shí)驗(yàn)分 溶栓無超聲無微泡 單純微泡 單純超聲組（占空比 單純超聲組（占空比 單純超聲組（占空比 單純超聲組（占空比 聲組與超聲+微泡組比較（占空比分別為0.216%、1.080%、3.600%、7.200%）P<0.01,超聲+微泡組（占空比分別為0.216%、1.080%、3.600%、7.200%）組間比較P<0.01。0 溶栓率百分比圖2 0 溶栓率百分比 （7.200%，溶栓率比較（X±S，n=8） 無超聲無微泡 單純微泡 單純超聲組（聲壓1檔 單純超聲組（聲壓3檔 單純超聲組（聲壓6檔 超聲+微泡組（聲壓1檔 超聲+微泡組（聲壓3檔 超聲+微泡組（聲壓6檔 注：無超聲無微泡組與微泡組比較，P>0.05；無超聲無微溶溶栓率百分比（7.200%678不同（7.200%678不同聲壓條件下二、病理學(xué)檢查(例圖1)；單純微泡治療后，血凝塊中紅細(xì)胞分布仍較均勻致密，只在討血栓形成和栓塞是急性心肌梗死（AMI）、腦卒中、肺栓塞等疾病的主DattaS等[1]進(jìn)行的體外實(shí)驗(yàn)結(jié)果表明超聲具有溶栓能力，并且超聲的溶栓能力與流效應(yīng)等[1-7]。超聲主要是通過空化效應(yīng)和機(jī)械效應(yīng)對(duì)生急劇的破壞作用。這種空化現(xiàn)象可產(chǎn)生高達(dá)1～3個(gè)大氣壓的“內(nèi)”效應(yīng)，使比較P0.01,差異有非常顯著性意義，說明單純超聲治療對(duì)體外血栓是有明確溶解血栓的作用的認(rèn)識(shí)已經(jīng)有幾十年了。Philippe等[21]使用超聲頻率是20KHz,驗(yàn).Rosenshin等[22]0.5,超聲出聲壓設(shè)為055/cm2.mm[23]、Singh[24]等證明超聲對(duì)于急慢性動(dòng)脈栓塞的治療是安全和[17]聲空化效應(yīng)是指存（通常10w/m爆沖[25]。單純體外治療超聲組與體外治療超聲+微泡組溶栓率比較，P<0.01,非常顯著性意義，說明微泡有增強(qiáng)體外治療性超聲溶栓的作用.光鏡下結(jié)果顯示幾做pfl和olld等[26]形UnrE等[7]ui[28]18290]（2[46]。方式4（即治療4s間歇4s的交替治療方式，治療時(shí)間30min，輸出聲壓均為6檔Suhkova等[31]在他們的實(shí)驗(yàn)中將超聲輸出聲壓設(shè)定為1/cm2，改變占空比從1100%，結(jié)果發(fā)現(xiàn)隨著占空比的加大，溶栓率不斷地增高。on[32]、在實(shí)驗(yàn)二中，固定治療方式4（即治療4s間歇4s的交替治療方式，治療時(shí)效應(yīng)。SimonS[35]、Lauer[36]、Luo[37]、Suchkova等[31，38]]的實(shí)驗(yàn)結(jié)果也表明，超聲溶栓效應(yīng)與聲壓呈正相關(guān)。嚴(yán)碧歌[39]、Schafer等[40]在溶栓中改變超聲的各種結(jié)高微泡聯(lián)合超聲的助溶作用。本研究創(chuàng)新性的自我評(píng)價(jià)DattaS,CoussiosCC,McAdoryLEetal.Correlationofcavitationwithultrasoundenhancementofthrombolysis.UltrasoundMedBiol2006;32:1257-1267.FrenkelV,OberoiJ,StoneMJ,etal.Pulsedhigh-intensityfocusedultrasoundenhancesthromblysisinaninvitromodel.Radiology，2006，239:86-93.BirnbaumY,LuoH,NagaiT,etal.Noninvasiveinvivoclotdissolutionwithoutthrombolyticdrug:recizationofthrombosediliofemoralarteriesbytranscutaneousultrasoundcombinedwithintravenousinfusionofmicrobubbles.Circulation1998;97:130-134.PolakJF.Ultrasoundenergyandthedissolutionofthrombus.NEnglJMed，HohmannJ,AlbrechtT,HoffmannCW,etal.Ultrasonographicdetectionoffocalliverlesions:increasedsensitivityandspecificitywithmicrobubblecontrastagents.EurJRadiol，2003，46(2):147-59.XieF,TsutsuiJM,LofJ,etal.Effectivenessoflipidmicrobubblesandindeclottingthrombosis.UltrasoundMedBiol2005;31:979-SakharovDV,HekkenbergRT,RijkenDC.Accelerationoffibrinolysisbyhighfrequencyultrasound:Thecontributionofaccousticstreamingandtemperaturerise.ThrombRes，2000，100:333-340.DoffelY，WermkeW.Neuroendocrinetumors:characterizationcontrast-enhancedultrasonography.Ultraschallmed，2008，29(5):506-PorterTR,LeVeenRF,FoxR,etal.Thrombolyticenhancementwithperfluorocarbonexposedsonicateddextrosealbuminmicrobubbles.AmHeartJ,MizushigeK,Kon.OhmoriK,etal.Enhancementofultrasound-acceleratedthrombolysisbyechocontrastagents:dependenceonmicrobubblestructure.UltrasoundMedBiol,1999,25(9):1431-1437.pietersM,HekenbergRT,BarrettBM,etal.TheEffectof40KHZultrasoundtissue-plasminogenactivator-inducedclotlysisinthreeinvitromodels.UltrasoundMedBiol,2004,30:1545-1552.TachibanaK,TachibanaS.Albuminmicrobubbleecho-contrastmaterialasanenchancerforultrasoundacceleratedthrombolysis.Circulation，1995，NedelmannM,EickeBM,NolleF,etal.TheultrasoundcontrastmediumLevovistincreasesthethrombolyticeffectoflowfrequencyultrasound.Med.Klin，SunQF,DuF,XuY,etal.Ultrasound-mediatedmicrobubbleenhancesbonemorphogeneticprotein-2geneexpressioninmouseskeletalmuscles.HuaXiKouqiangYiXueZaZhi，2009，27(1):84-7.VonHerbayA,BarreirosAP,IgneeA,etal.Contrast-enhancedwithSonovue:differentiationbetweenbenignandmalignantlesionsofthespleen.UltrasoundMed，2009，28(4):421-34..4CulpWC,MccowanTC.Ultrasoundaugmentedthrombolysis.CurrMedImag8（7：425HollandCK,ApfelRE.Thresholdsfortransientcavitationproductionbypulsedultrasoundinacontrollednucleienvironment.JAcoustSocAm，1990，ThomasRPorter,FengXie.Ultrasound,microbble,andthrombolysis[J].ProgressinCardiovascularDiseases,2001,44(2):101-110.WinterPM,ShuklaHP,CaruthersSD,etal.Molecularimagingofhumanthrombuswithcomputedtomography.AcadRadiol2005;12(Suppl1):S9-S13.MolinaCA,AlexandrovAV,DemchukAM,etal.Improvingthepredictiveaccuracyofrecizationonstroke 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只在表面形成小的散在空隙（HE染色×40）。 例圖3.單純ETUS（聲壓固定為6檔）治療后,由于紅細(xì)胞的破壞，紅細(xì)胞 例圖4.ETUS+MUCA（聲壓固定為6檔）治療后,由于紅細(xì)胞的破壞，紅細(xì)胞間出現(xiàn)了不同程度的缺損，血栓纖維網(wǎng)架結(jié)構(gòu)不同程度破壞.A(占空比為C(占空比為3.600%):血栓塊紅細(xì)胞數(shù)量減少更加明顯，紅細(xì)胞結(jié)構(gòu)分散， C例圖5單純ETUS（固定占空比為7.200%）治療后,由于紅細(xì)胞的破壞，紅細(xì)胞間出現(xiàn)了不同程度的缺損.A(聲壓為1檔):紅細(xì)胞間出現(xiàn)了小缺損;B(聲壓為3檔）,紅×40 C例圖6.ETUS+MUCA（占空比為7.200%）治療后,由于紅細(xì)胞的破壞，紅細(xì)胞間出現(xiàn)了不同程度的缺損，血栓纖維網(wǎng)架結(jié)構(gòu)不同程度破壞.A(聲壓為1檔):血維網(wǎng)架稍有破壞;B(聲壓為3檔）,血栓塊紅細(xì)胞數(shù)量減少，紅細(xì)胞結(jié)構(gòu)稍分塊紅細(xì)胞數(shù)量減少更加明顯，紅細(xì)胞結(jié)構(gòu)分散，紅細(xì)胞間出現(xiàn)了大缺損，綜一、微泡超聲造影劑在和治療方面的研究現(xiàn)自1968年Gramiak首次了微泡超聲造影劑（MicrobubbleUltrasoundU靶向轉(zhuǎn)染、突破生物學(xué)屏障等方面，具有廣闊的應(yīng)用前景。目前，UA在治療方面的運(yùn)用已成為超聲醫(yī)學(xué)研究的熱點(diǎn)。二、超聲造影劑的發(fā)展ueOptiso造影劑穩(wěn)定性強(qiáng)，主要是因?yàn)槲⑴萃鈿ぞ哂袕椥浴㈨g度大，其的氣體是低彌泡，內(nèi)含氣體，外周穩(wěn)定性好的外膜。靶向微泡表面附著有針對(duì)靶部位的特三、超聲微泡造影劑在治療方面的運(yùn)用（AMI6小時(shí)的患者溶栓效果不理想。超聲溶栓是20世紀(jì)90年代發(fā)展起來的一項(xiàng)新技術(shù)，酶[815]氣內(nèi)炸裂小超[1617]Port等用20z，1.5/m的超聲照射輸入尿激酶（）的體外實(shí)驗(yàn)，同時(shí)注入PS微泡，其結(jié)果顯示在有微泡時(shí)，超聲能使的溶栓效果增加1.5倍至3倍，甚至可以在不使用溶栓劑的情況下使血凝塊溶解[10]。研究表明[11]：研究表明：超聲空化效應(yīng)的發(fā)生是一種閾過程，空[815]一系列的研究發(fā)現(xiàn)，超聲造影劑可成為安全、有效、簡(jiǎn)便的靶向轉(zhuǎn)染載體 達(dá)露DNA的水平提高300倍[20]。內(nèi)小鼠動(dòng)物實(shí)驗(yàn)亦證明，內(nèi)注射露增強(qiáng)1000倍[21]。超聲造影劑靶向運(yùn)送有望成為一種安全、高效、無創(chuàng)的要發(fā)病原因。Song等[22]于2002年首次通過實(shí)驗(yàn)證明，超聲破壞微泡可促進(jìn)血管[23]。微泡在腫[24][25]。超聲破壞微泡產(chǎn)生的生物效應(yīng)可使腫瘤新生血管發(fā)生明顯的超微結(jié)構(gòu)改變四、超聲微泡造影劑存在問題穩(wěn)定性方面：攜藥物或治療的微泡造影劑應(yīng)具備很強(qiáng)的穩(wěn)定性才能到[28,29]，使細(xì)胞膜通透性[31-34]五、超聲微泡造影劑的發(fā)展前景攜帶治療藥物或的靶向微泡是今后超聲造影研究的努力方向。隨著分子 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