摘要
目的优化培养乳鼠心肌细胞的技术,筛选出简便、准确、特异的提取心肌细胞β3-肾上腺素能受体(β3-AR)膜蛋白的方法。方法使用Ⅱ型胶原酶和差速贴壁法收集心肌细胞,分别用总蛋白法、超速离心法、试剂盒法提取心肌细胞β3-AR膜蛋白;BCA法进行蛋白定量;Western blot法检测蛋白样品中β3-AR和GAPDH相对含量及提取蛋白的特异性。结果优化心肌细胞的培养方法可以使所得细胞产量大、浓度高,满足后续实验。试剂盒法提取蛋白的浓度(8.26±0.29)g/L>总蛋白法提取法的(5.12±0.47)g/L>超速离心法的(3.20±0.37)g/L,差异均有统计学意义。Western blot检测结果示,试剂盒提取法所得β3-AR膜蛋白含量(0.22±0.05)>超速离心法(0.09±0.03)>总蛋白提取法(0.01±0.01),差异均有统计学意义。结论优化培养心肌细胞的方法可获得高产量、高纯度的细胞。试剂盒提取法可有效提高β3-AR膜蛋白的浓度和特异性。
Objective To optimize primary cultures techniques of isolating neonatal rat cardiomyocytes and to com-pare three different methods of extractingβ3-adrenergic receptor(β3-AR)membrane protein from cultured neonatal rat car-diomyocytes. Methods TypeⅡcollagen and differential velocity adhesion were used to collect primary cardiomyocytes. To-tal protein method, ultracentrifugation method, extract kit method were used to isolate cardiomyocytesβ3-AR membrane pro-teins. The BCA method was applied for protein quantification. Relative content ofβ3-AR membrane protein and GADPH in the sample were examined by western blot. Results Optimizing culture and isolation skills can produce a great quantity of cardiomyocytes in high concentration.The kit method acquired a higher level of protein concentration(8.26±0.29)g/L than to-tal protein method(5.12±0.47)g/L does than ultracentrifugation method(3.20±0.37)g/L does all of which were with signifi-cant difference(P 〈 0.05). The concentration of β3-AR membrane protein was higher if obtained by kit method(0.22 ± 0.05)than ultracentrifugation method(0.09 ± 0.03)than total protein method (0.01 ± 0.01) with significant difference(P 〈0.05). Conclusion optimizing methodology can obtain abundant myocardial cells in high concentraion. The kit method of isolating primary culturedβ3-AR membrane proteins result in improved concentration and specificity of membrane protein.
出处
《天津医药》
CAS
2015年第6期599-602,共4页
Tianjin Medical Journal
基金
国家自然科学基金-地区科学基金项目(81260028)
石河子大学科学技术研究发展计划"自然科学与技术创新"团队创新项目(2011ZRKXTD-07)