CAJ | 학술논문

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。
목적：우화배양유서심기세포적기술，사선출간편、준학、특이적제취심기세포β3-신상선소능수체（β3-AR）막단백적방법。방법사용Ⅱ형효원매화차속첩벽법수집심기세포，분별용총단백법、초속리심법、시제합법제취심기세포β3-AR막단백；BCA법진행단백정량；Western blot법검측단백양품중β3-AR화GAPDH상대함량급제취단백적특이성。결과우화심기세포적배양방법가이사소득세포산량대、농도고，만족후속실험。시제합법제취단백적농도（8.26±0.29）g/L＞총단백법제취법적（5.12±0.47）g/L＞초속리심법적（3.20±0.37）g/L，차이균유통계학의의。Western blot검측결과시，시제합제취법소득β3-AR막단백함량（0.22±0.05）＞초속리심법（0.09±0.03）＞총단백제취법（0.01±0.01），차이균유통계학의의。결론우화배양심기세포적방법가획득고산량、고순도적세포。시제합제취법가유효제고β3-AR막단백적농도화특이성。
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.