天津医药

天津医药 ›› 2015, Vol. 43 ›› Issue (6): 599-602.doi: 10.11958/j.issn.0253-9896.2015.06.006

• 实验研究 • 上一篇    下一篇

心肌细胞的培养鉴定及其β3-AR蛋白提取方法的比较

马苗苗 1， 胡小芳 1， 朱晓丽 1， 王丽 1， 马依彤 2， 杨毅宁 2， 陈邦党 2   

  1. 1新疆石河子大学医学院第一附属医院心内科 ； 2新疆医科大学第一附属医院心脏中心
  • 收稿日期:2014-11-06 修回日期:2015-01-25 出版日期:2015-06-15 发布日期:2015-06-10
  • 通讯作者: 王丽 E-mail:mcmwl@163.com
  • 基金资助:
    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）

Cultured cardiomyocytes identificaiton and different methods of extracting β3-AR membrane#br# protein comparison

MA Miaomiao1, HU Xiaofang1, ZHU Xiaoli1, WANG Li1, MA Yitong2, YANG Yining2, CHEN Bangdang2   

  1. 1 Department of Cardiology， the First Affiliated Hospital, School of Medicine, Shihezi University, Shihezi 832008， China;
    2 Heart Centre, The First Affiliated Hospital of Xinjiang Medical University

  • Received:2014-11-06 Revised:2015-01-25 Published:2015-06-15 Online:2015-06-10

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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。

关键词: 受体,肾上腺素能β3, 肌细胞, 心脏, 提取法, 细胞培养技术, 心肌细胞, β3-AR 膜蛋白

Abstract: Objective To optimize primary cultures techniques of isolating neonatal rat cardiomyocytes and to compare three different methods of extracting β3-adrenergic receptor（β3-AR）membrane protein from cultured neonatal rat cardiomyocytes. Methods TypeⅡ collagen and differential velocity adhesion were used to collect primary cardiomyocytes. Total protein method, ultracentrifugation method, extract kit method were used to isolate cardiomyocytes β3-AR membrane proteins. The BCA method was applied for protein quantification. Relative content of β3-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 total protein method（5.12±0.47） g/L does than ultracentrifugation method（3.20±0.37） g/L does all of which were with significant 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 ofisolating primary cultured β3-AR membrane proteins result in improved concentration and specificity of membrane protein.

Key words: eceptors, adrenergic, beta-3, myocytes, cardiac, extraction, cell culture techniques, cardiomyocytes, β3-
AR membrane protein