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应用胰蛋白酶分次消化法分离乳鼠心肌细胞,以差速贴壁法纯化心肌细胞,α-sarconme-actin抗体免疫细胞化学染色鉴定心肌细胞。心肌细胞在无血清无酚红培养基中培养48h后,用双氢睾酮(DHT)诱导心肌细胞肥大,建立心肌细胞肥大模型。24h后检测心肌细胞肥大的指标心肌细胞表面积;BCA法测定心肌细胞蛋白含量;半定量RTPCR两步法检测心肌细胞肥大的特征性基因—心房利钠因子(atrial natriuretic factor,ANF,β-肌球蛋白重链(β-myosin heavy chain,β-MHC)mRNA的表达。结果显示免疫细胞化学染色显示培养的心肌细胞纯度达到90%以上,心肌细胞分离良好。与对照组相比,10-8 mol/L的DHT能显著的增加心肌细胞表面积、蛋白质含量、ANP和β-MHC基因表达的增加(P<0.01),心肌细胞肥大模型建立成功。
Cardiomyocytes were isolated by trypsin digestion and cardiomyocytes were purified by differential adherence. Cardiomyocytes were identified by immunocytochemical staining with α-sarconme-actin antibody. Cardiomyocytes were cultured in serum-free phenol red-free medium for 48 h, and then induced by DHT to induce cardiomyocyte hypertrophy. Cardiomyocyte hypertrophy model was established. The cardiomyocyte surface area was measured after 24 h, the protein content of cardiomyocytes was measured by BCA method, the gene of atrial natriuretic factor (ANF), β-myosin The results of immunocytochemical staining showed that the purity of cardiomyocytes was above 90% and the cardiomyocytes were well separated.Compared with the control group, the expression of β-MHC mRNA in 10-8 mol / L of DHT can significantly increase myocardial cell surface area, protein content, ANP and β-MHC gene expression increased (P <0.01), myocardial cell hypertrophy model was established.