胎鼠肺泡ⅱ型上皮细胞的分离及原代培养

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1、胎鼠肺泡Ⅱ型上皮细胞的分离及原代培养付红敏，许峰，黄波，杨鸣，符跃强，方芳，匡凤梧（重庆医科大学儿童医院PICU，重庆400014）摘要：目的探讨胎鼠肺泡Ⅱ型上皮细胞（AECⅡ）的分离、纯化及原代培养方法，为有关胎儿肺发育及新生儿肺部疾病的研究奠定基础。方法采用胰酶结合胶原酶的消化方法，分离肺组织细胞成份，然后经差速离心和差速贴壁的方法纯化AECⅡ，进行原代培养；通过台盼蓝染色检测细胞活力，倒置相差显微镜观察细胞生长特点及形态特征，透射电镜鉴定，改良巴氏染色检测细胞纯度以及免疫荧光技术检测表面蛋白C（SPC）的表达。结果每3～5只胎鼠可获得AECⅡ(36±5)

2、×106，活力(98±2)％。镜下观察原代AECⅡ呈岛状生长，外观呈多边形或立方形。透射电镜可见特征性的板层小体，改良巴氏染色见胞质内有较多颗粒，纯度为(96±3)％，呈现SPC绿色免疫荧光的细胞占96％以上。结论利用胰酶和胶原酶消化，以及差速离心和差速贴壁的方法可成功分离出高产量、高纯度的胎鼠AECⅡ，能满足体外进一步实验的需要。关键词：胎鼠；肺泡Ⅱ型上皮细胞；原代培养；分离中图分类号：R714.5；R714.7；Q813.1IsolationandprimarycultureoftypeⅡalveolarepithelialcellsfromfetalra

3、tlungFUHong-min,XUFeng,HUANGBo,YANGmin,FUyueqiang,FANGFang,KUANGFeng-wu.PediatricsIntensiveCareUnit,Children’sHospital,ChongqingUniversityofMedicalSciences,Chongqing400014,China【Abstract】ObjectiveTodeveloptheisolationandpurificationtechnologyfortypeⅡalveolarepithelialcells(AECⅡ)offe

4、talrat,thusprovidingexperimentalmeansforthe基金项目：国家自然科学基金面上项目(基金号：30670931)NationalNaturalScienceFoundation(NO.30670931)高等学校博士学科点专项科研基金项目（基金号：20070631010）ResearchFundfortheDoctoralProgramofHigherEducation(NO.200706310109)studyoflungdevelopmentorneonatallungdisease.MethodsLungtissueso

5、f19-dayfetalratsweredigestedwithtrypsinandcollagenase,thenpurifiedforAECⅡwithdifferentcentrifugalforceandrepeatedattachment.Cellviabilitywasevaluatedbytypraninclusiondyingbeforeplatingintosix-wellflask.Growthstatusandshapeofattachedcellswereobservedwithinvertedphasecontrastmicroscop

6、e.AECⅡwereidentifiedbyelectronmicroscopeanditspercentagewasassessedbymodifiedPapanicolaoudyingandimmunofluorescence,thelatteraimingtodetectexpressionofsurfactantproteinC(SPC)inAECⅡ.ResultsThetotalamountofcellsweharvestedfrom3to5fetalratwas(36±5)×106withaviabilityof(98±2)％.Cellswerel

7、ikepolygonalandconnectedlikeislandundermicroscope．AECⅡwasascertainedbylamellarbodiesfoundincytoplasmaunderelectronmicroscopeanditspercentagewas(96±3)％identifiedbymodifiedPapanicolaoudying,thesameastheresultbyimmunofluorescenceforSPC.ConclusionHighlyPurifiedandviableAECⅡcouldbeachiev

8、edbyourmethodsandth