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1、成年兔心耳肌细胞的分离标记与自体移植张浩，蔡振杰，俞世强，赵璧君，张近宝【关键词】心肌/细胞学Isolation,labellingandautotransplantationofleftauriclemyocytesinadultrabbit 【Abstract】AIM:Todevelopareliablemethodforisolationandfluorescentlabelingofadultrabbitleftauriclemyocytesandtoinvestigatethesurvivalofgraftedautolog
2、ousleftauriclemyocytes.METHODS:Tlyassignedtotransplantgroupandcontrolgroup(n=10).Theleftauricleofrabbitedium(controlgroup)alleftventricularanteriorensethods.RESULTS:Mostoftheisolatedcellsorphologicfeatureofthesecellscorrespondedtothatofauriclecardiomyocytesandthecellacti
3、vityyocardialinfarction(MI)areaoftransplantgroup,andthenucleiedthesurvivalofimplantedcellsandthereliabilityofDAPIlabeling.VasculardensityintransplantgroupedigestionandDAPIlabelingarereliablemethodsfortheisolationandlabelingofleftauriclemyocytesinadultrabbits.IsolatedandD
4、APIlabeledauriclemyocytescansurviveafterautograftedintonormalventricularanteriorayimproveperipheralvascularproliferation. 【Keyyocardium/cytology;isolation;label;transplantation,autologous 【摘要】目的：建立可靠的成年兔心耳心肌细胞的急性分离和荧光标记的方法，观察心耳肌细胞移植到自体左室前壁的存活状况.方法：成年新西兰白兔20只，随机分为移植组和对照
5、组（n=10）.结扎并剪取自体左心耳组织，急性消化分离为单细胞，经DAPI标记后分别将细胞悬液和培养基注射到移植组和对照组自体正常左室前壁内.4a公司）,4,6二脒基2苯茚二酮(Sigma公司). 1.2方法 1.2.1自体左心耳心肌组织的取材取成年兔经耳缘静脉注射戊巴比妥钠（30mg/kg），左侧胸骨旁切口，切断第4肋骨进胸，向上推开胸腺，剪开心包，显露并牵引左心耳，用4号丝线结扎左心耳基底部后，剪取左心耳并立即置于预冷（4℃）的普通台式液中.用盐水纱布覆盖伤口. 1.2.2左心耳心肌细胞的消化分离参照文献〔2〕，在无钙台式液(
6、mol/L:NaCl140,KCl5.4,MgCl21,酶糖10,HEPES5,pH7.4)中洗去血凝块，用眼科剪将心耳组织剪碎，移入含有1.25g/L胰酶、0.25g/L胶原酶的无钙台式液中，37℃消化10min.轻柔吹打，自然沉淀后弃上清.再加入酶消化10min，轻柔吹打后，吸取上清，移入等体积含100mL/L新生牛血清的DMEM培养液中.重复消化剩余组织2～3次，直至基本不可见有形组织.将收集的细胞悬液1000r/min,离心5min,弃上清，调整细胞密度为2×109/L. 1.2.3分离细胞的活性鉴定 1.2.7结果观察4a
7、npediatricandadultventricularmyocytesinculture:AssessmentofphenotypicchangesanJ,DuongM,ZibaitisA,etal.Myocardialtissueengineeringyoblastimplantation〔J〕.JThoracCardiovascSurg,1998;116:744-751. 〔5〕LiRK,JiaZQ,alhotraA,LiBS,etal.Effectsofconstitutiveoverexpressionofinsulin2l
8、ikegroechanicalcharacteristicsandmolecularpropertiesofventricularmyocytes〔J〕.CircRes,1998;82:594-603.
