摘要
目的建立新生小鼠心肌细胞（CM）的分离纯化、培养及鉴定的方法。方法采用酶消化法分离消化心室组织,差速贴壁法和化学方法纯化CM;培养7d后,利用免疫荧光技术和RT-PCR技术检测部分CM特异基因在蛋白和mRNA水平的表达;改进透射电镜的常规操作方法,观察CM超微结构。结果采用0.08%胰蛋白酶和0.04%胶原酶Ⅱ消化心室组织后,细胞成活率为98%,贴壁培养第3d出现同簇细胞的同步跳动;抗心肌肌钙蛋白（cTnT）免疫荧光染色阳性率为99.07%;以骨髓间充干细胞为阴性对照,RT-PCR结果显示CMα和β两型心肌肌球蛋白重链、cTnT基因表达呈阳性;经3%~4%琼脂预包埋法处理可以对少量细胞进行超薄切片的制作,CM具有正常的超微结构。结论建立了新生小鼠CM分离纯化培养和鉴定方法。
Objective To discuss the methods of separation,purify and culture of neonatal mice cardiomyocytes（CM） for inducing mesenchymal stem cells（MSCs） to CM by coculture,and found the way to identify the cells.Methods CM from ventricular tissues of neonatal mice were separated with enzyme digestion,purified by differential and adherent,and treated with 5-bromodeoxyuridine（5-Brdu）.The expressions of CM mRNA and protein were detected with reverse transcription-polymerase chain reaction（RT-PCR） and immunofluorescence staining,and the ultras-tructural characteristics of CM were observed with transmission electron microscope after culture for 7 days.Results CM with well morphology and spontaneous beating obtained by the use of 0.08% trypsin and 0.04% collagenase type Ⅱ,98 % cells are alive,and then CM were arranged in loose and jumping synchronously by adherent culture for 3 days.The percentage of positive cell（CM） were 99.07%,marked with anti-cardiac Troponin T（anti-cTnT） antibody by immuofluorescence staining.The expression of myocardial myosin heavy chain α/β（MHC α/β）,cTnT gene in CM were detected apparently by RT-PCR with MSCs as negative control detetednegative control.The Ultra-structure of few CM can be observed,used 3%-4% Agar to pre-embed and then got the extra-thins lice.Conclusions Our studies found a way to obtain and identify neonatal mouse CM with nomal tructure and function,and provide a foundation for further inducing MSCs by cocultrue with CM,and identifying the cells after differentiation.
出处
《中国医药指南》
2012年第26期20-23,共4页
Guide of China Medicine
