构建原代分离培养与鉴定小鼠肺泡Ⅱ型上皮细胞的方法及模型VIP免费

中国组织工程研究与临床康复第74誊第15嬲2010—04—09出版JournalofClinicalRehabilitativeTissueEngineeringResearchApril9，2010VoL14，No．15构建原代分离培养与鉴定小鼠肺泡II型上皮细胞的方法及模型：l：：|：☆郑金旭，黄振杰，汤艳，丁明《辎％槲№《目螭∞赫Ⅻ目目∞#∞黼《$目黼黼熵m$#Ⅻ∞∞Ⅻ∞《《Ⅻ∞《踹∞蛳Ⅻ％㈣㈣@∞Ⅻ№Ⅻ#∞∞械№#镕剿№@黼Ⅻ《黼ⅫⅫ№∞∞蛹∞∞#@ⅫⅫmW㈥∞W酣∞≈$∞Ⅻ∞酬％Ⅻ№@@Ⅻ∞滞∞《№‰∞@姆Ⅻ㈣№№Ⅻ∞Ⅻ$Ⅻ∞㈣Ⅻ㈣ⅫⅫ∞∞∞∞《ⅫWWN嘲镕㈣㈣Ⅻ斓鲫《Ⅻ@∞《瓣Ⅻ㈣∞∞《磷№*‰猕Isolation，primarycultureandidentificationoftype11alveolarepithelialcellsfrommiceZhengJin-xu，HuangZhen_jie，TangYan，DingMingAbstractBACKGROUND：TypeIIalveolarepithelialcells(AECII)playanimportantroleinlungdevelopmentandregulationof．1ungfunction．1tiscloselyrelatedwiththegenesisanddevelopmentofpulmonaryfibrosis．1ungcancerandsoon．1nordertomakean『仃vitrostudyofthesediseases．astableandeffectivecelImodeIforisolation．primarycultureandidentificationofAECIIshouldbeestablished．OBJECTlVE：TOdevelopareliablemethodfortheisolation，purification，primarycultureandidentificationofAECIIfrOmmice，andprovideafoundationforthefurtherstudyofpulmonaryfibrosisandlungcancer．METHODS：①Thelungtissueofmicewasdigestedwithlow-concentrationtrypsinandcollagenaseI．Thatis，lungtissuesofmicewerecutintosmallpieces，digestedwithtrypsin，andterminatedthedigestionbyaddingDMEMcontainingfetalbovineserum．Thecellsweredigestedonceagainusingtrypsin．andaddingcollagenaseIaftertermination．(函AEC11waspurifiedbydifferentialcentrifugationandimmuneadherenceforprimaryculture．ThepneumocytessuspensionwasincubatedinIgGcultureplate，andliquidcontainingnon-adherentcellswereonceagainculturedinIgGcultureplate，thensupernatantwereabandoned，andthecellswereculturedjnculturedishandcultureplate．ThemorphologyandgrowthcharacteristicsofprimarilyculturedAEC11wereobservedbyanjnvertedmicroscope；theyield．purityandviabilityofAEC11weredetermined．AEC11wasidentifiedbyimmunocytochemicalstainingsudactantapoproteinA(SP-A)andSP-C，andthespecificcelIstructurewasobservedunderatransmissionelectronmicroscope．RESULTSANDCONCLUSlON：Bythismethod。anamountof(4．8+1．2)x105AEC11wereharvestedfromeachmousewithapurityof(85土2．4)％andaviabilityof(92±2．4)％．Underaninvertedmicroscope，AECIIinprimarycultureshowedashapeofroundorcubeandanisland—likegrowth．ThejmmunocytochemistryshowedthatthecytoplasmpresentedbuffySP—AandgreenSP—C．TypicaIfeaturesofAECIIincludingIamellarbodiesandmicrovillicouldbeseenunderatransmissionelectronmicroscope．Theresultsdemonstratedthat，AECIrofgreatamountandhighpuritycouldbeobtainedbythismethod，whichmeetstherequirementofacellmodelestablishingforfurther／nvitrostudy．ZhengJX，HuangZJ，TangY，DingM．Isolation，primarycultureandidentificationoftypeIIalveolarepithelialcellsfrommice．ZhongguoZuzhiGongchengYanjiuyuLinchuangKangfu．2010；14(151：2761—2764．【http：／／www．crter．cnhttp：／／en．zglckf．corn】摘要背景：肺泡II型上皮细胞在肺发育和肺功能调节中起重要作用，它与肺纤维化和肺癌等疾病的发生发展有密切联系，为了对这些疾病进行体外实验研究，必须对肺泡II型上皮细胞进行分离、原代培养与鉴定以建立一个稳定高效的细胞模型。目的：建立一套可靠的小鼠肺泡II型上皮细胞分离、纯化、原代培养与鉴定的方法，为肺纤维化及肺癌等疾病的进一步研究奠定基础。方法：①用低浓度胰酶联合I型胶原酶消化法，分离小鼠肺组织细胞成分。取出小鼠肺组织，剪成小块...