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目的观察生后不同时间乳鼠心肌细胞的形态结构和功能特征,建立理想的分离和纯化乳鼠心肌细胞技术。方法采用胰蛋白酶消化和机械分离,结合两次差速贴壁法以及使用溴脱氧尿嘧啶核苷(BrdU),从生后1~7d乳鼠心脏分离和纯化心肌细胞。观察细胞的形态和自发性搏动,并用心肌特异性肌钙蛋白丁(cTnT)免疫细胞化学染色进行鉴定。利用原位透射电镜术观察细胞超微结构,并观察其对肾上腺素和异丙肾上腺素的反应。结果从乳鼠心脏分离的细胞95%以上为心肌细胞,存活率为95%以上。乳鼠心肌细胞包括短柱状或杆状细胞和不规则形细胞。生后1~3d鼠大部分杆状细胞呈现幼稚心肌细胞的超微结构特征。6~7d鼠的杆状细胞具有类似于成熟心肌细胞的超微结构特征,cTnT呈高表达,横纹明显。不规则形细胞含有少量肌丝束,排列不规则。少数体积较小的细胞呈现未分化细胞的超微结构特征。培养72h后80%以上的细胞出现自发性搏动。肾上腺素和异丙肾上腺素刺激后,搏动细胞数目增多,搏动增强。生后6~7d鼠杆状细胞的药物反应较强。结论两类心肌细胞的超微结构特征、自发性搏动和对肾上腺素和异丙肾上腺素的反应不同。生后6~7d鼠心肌细胞较适合于成熟心肌细胞的体外实验研究。本实验建立的方法是一种较理想的乳鼠心肌细胞培养方法。
Objective To observe the morphological structure and functional characteristics of neonatal rat cardiomyocytes at different time points after birth and to establish an ideal technique for the isolation and purification of neonatal rat cardiomyocytes. Methods Cardiomyocytes were isolated and purified from the heart of 1 ~ 7 d postnatal rats by trypsin digestion and mechanical separation combined with two differential adherent methods and using bromodeoxyuridine (BrdU). Cell morphology and spontaneous beating were observed and identified by cTnT immunocytochemistry. The in situ transmission electron microscopy was used to observe the ultrastructure of the cells and observe its response to epinephrine and epinephrine. RESULTS: More than 95% of cells isolated from the heart of a neonatal rat were cardiomyocytes with a survival rate of 95% or more. Neonatal cardiomyocytes include short columnar or rod-shaped cells and irregularly shaped cells. Most postnatal 1 ~ 3d rat rod-shaped cells showed ultrastructural features of naïve cardiomyocytes. 6 ~ 7d rat rod cells have similar ultrastructural features of mature cardiomyocytes, cTnT was highly expressed stripes obvious. Irregular cells contain a small amount of myofilament, arranged irregularly. A small number of smaller cells present the ultrastructural features of undifferentiated cells. More than 80% of cells cultured for 72 hours showed spontaneous beats. After stimulation with epinephrine and isoprenaline, the number of beating cells increased and the beats increased. 6 ~ 7d after birth, rat rod-shaped cells stronger drug response. Conclusion The ultrastructural features, spontaneous pulsation and response to epinephrine and epinephrine differ between the two types of cardiomyocytes. 6 ~ 7d after birth, rat cardiomyocytes more suitable for mature cardiomyocytes in vitro study. The experiment established method is a more ideal method of neonatal rat cardiomyocyte culture.