资源描述:
《肺成纤维细胞分离》由会员上传分享,免费在线阅读,更多相关内容在教育资源-天天文库。
1、【摘要】目的:建立一套可靠的鼠肺细胞分离、纯化和培养技术,用于体外研究间质性肺病的发病机制及治疗方法。方法:采用胰酶消化出生后2dSD大鼠肺组织块及SD大鼠肺泡灌洗液,经差时贴壁法,分离、纯化红皮鼠肺成纤维细胞(lungfibroblast,LF)和SD大鼠肺泡巨噬细胞(pulmonaryalveolarmacrophage,AM),并进行原代培养。用波形蛋白(Vimentin)、CD14细胞免疫荧光染色、蛋白质印迹和电镜对所分离的细胞进行鉴定。结果:所得细胞产量大、纯度高。细胞免疫荧光染色LF细胞波形
2、蛋白染色阳性而CD14染色阴性;AM细胞则相反。扫描电镜观察可见肺成纤维细胞有微绒毛,细胞间连接成网状,蛋白质印迹结果显示波形蛋白表达。结论:该方法是一种可靠的红皮鼠肺成纤维细胞及大鼠肺巨噬细胞的分离纯化培养技术,可用于间质性肺病(interstitiallungdisease,ILD)的发病机制及治疗方法的体外研究。【关键词】大鼠; 肺成纤维细胞; 巨噬细胞; 原代细胞培养[基金项目]江苏省高校自然科学研究计划项目(03KJD320076);镇江市社会发展科技计划项目(SH2004043) Is
3、olationandpurificationandprimarycultureoflungcellsfromrats YANYu-lan,LIUYang,BUXue-feng,ZHANGZhi-jian,ZHENGJin-xu (DepartmentofRespiratoryMedicine,theAffiliatedPeople′sHospitalofJiangsuUniversity,ZhenjiangJiangsu212002;SchoolofMedicine,JiangsuUniversit
4、y,ZhenjiangJiangsu212013,China) [Abstract]Objective:Todevelopeareliablemethodforisolation,purificationandprimarycultureoflungfibroblast(LF)andpulmonaryalveolarmacrophage(AM)fromratforstudiesonpathogenesisandtreatmentofinterstitiallungdiseaseinvitro.Meth
5、ods:Thelungtissueof2daysoldimmatureratswasdigestedwithtrypsin.LFsandAMswereisolatedandpurified,thencultured.ThenatureofthecultureswasidentifiedbyCD14staining,vimentinstaining,electronmicrographyandWesternblot.Results:Excellentyieldsofhighlypurified,cultu
6、rableAMsandLFscouldbeobtainedfromadultratsand2daybabyishratlungs,respectively.TheexpressionofCD14inAMswaspositiveandthatofvimentinnegativebyimmunofluorescencechemistry.Those,however,inLFswerejustopposite.RamifiedstracturebetweenpurifiedLFswereobservedbyu
7、ltrastructuralexaminationunderelectronmicrography.VimentininpurifiedLFswasrevealedbyWesternblot.Conclusion:Thistechniquemightbeareliablemethodforisolationandpurificationandprimarycultureofpulmonaryalveolarmacrophagesandlungfibroblastsfromratlungandcouldb
8、eusedforthestudyofinterstitiallungdisease [Keywords]lungfibroblasts;immaturerat;pulmonaryalveolarmacrophage;primarycellculture 原代培养又称初代培养,是自供体取得组织细胞后在体外进行的首次培养。原代培养的细胞来源于刚刚离体的组织和细胞,其生物学特性未发生很大变化,仍具有二倍体遗传特征,最接近和反映体内生长特性,对