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1、新生SD大鼠心肌細胞分離培養(yǎng)及純化的快速便捷方法及鑒定付微劉飛喬陸明張緒東牡丹江醫(yī)學(xué)院牡丹江市第二人民醫(yī)院摘要：R的探討一種體外培養(yǎng)存活率高、純度高、耗時少的心肌細胞改良方法。方法采用自配0.25%胰蛋白酶重復(fù)消化新生24h以內(nèi)乳鼠的心室組織多次，除第一次收集的上清外，其余收集到的細胞用含12%胎牛血清的高糖DMEM培養(yǎng)基中和以終止消化，差速貼壁90min，加入終濃度為0.lmmol/L的5-溴-2-脫氧尿苷(5-Brdu)純化心肌細胞。結(jié)果聯(lián)合應(yīng)用差速貼壁分離法和抑制成纖維細胞增殖的方法培養(yǎng)出的心肌細胞于倒置顯微鏡下觀察其細胞生長狀態(tài)良好，24h后基本
2、全部貼壁，心肌細胞呈梭形、三角形，不規(guī)則形等，搏動頻率在5(/00次/min,持續(xù)時間久，活力好且穩(wěn)定。經(jīng)心肌肌鈣蛋白免疫熒光鑒定心肌細胞純度為92.9%。結(jié)論木研究應(yīng)用改良的新生乳鼠心肌細胞培養(yǎng)方法，快速便捷，得到存活率較高、純度高、搏動狀態(tài)良好的心肌細胞，且培養(yǎng)耗吋短，可重復(fù)性高。可為研宂心臟疾病的基礎(chǔ)科學(xué)研宂提供理想的實驗?zāi)Ｐ?。關(guān)鍵詞：心肌細胞;免疫熒光;原代培養(yǎng);方法改良;快速便捷;作者簡介：付微(1991-),女，漢，在讀碩士，主要從事生理學(xué)研究，E-mail:943498398@qq.com。作者簡介：張緒東，教授，碩士生導(dǎo)師，E-mail:
3、81728836@qq.com。收稿日期：2017-08-19基金：黑龍江省創(chuàng)新訓(xùn)練項目(201510229014)ImprovementofPrimaryCardiomyocyteCultureinNewbornSDRatsFUWeiSchoolofBasicMedicalSciencesMudanjiangMedicalUniversity;Abstract：ObjectiveToexploreamethodforimprovingcardiomyocyteswithhighsurvivalrate,highpurityandlowtimeinvit
4、roculture.MethodsUsingself-contained0.25%trypsinrepeateddigestion24h24hneonatalventriculartissueseveraltimes,TheremainingcollectedcellswereneutralizedwithhighglucoseDMEMmediumcontaining12%fetalbovineserum,exceptforthefirstsupernatantcollectedTerminationofdigestion,differentialadh
5、erent90min,addingthefinalconcentrationof0.1mmol/L5-bromo-2-dcoxyuridine(5-Brdu)purificationofcardiomyocytes.ResultsThecardiomyocytesculturedunderthemethodofdifferentialadherenceseparationandinhibitionoffibroblastproliferationwereobservedunderinvertedmicroscope.Thecellswerewelladh
6、eredunder24h,andthemyocardiumwasspindle-shaped,triangular,irregularAndsoon,thepulsefrequencyof50-100times/min,lastingalongtime,goodvitalityandstability.Thepurityofmyocardialcellswas92.9%bycardiactroponinimmunofluorescence.ConclusionInthisstudy,themodifiedneonatalratcardiomyocytec
7、ulturemethodwasusedtoobtaincardiomyocyteswithhighsurvivalrate,highpurityandgoodpulsation,andthecultureisshortandreproducible.Canprovideanidealexperimentalmodelforthestudyofbasicscientificresearchonheartdisease.Keyword：Cardiomyocytes;jmmunof1uorescence;primaryculture;methodimprove
8、d;fastandconvenient;Received：2017-08-19隨
