CAJ | 학술논문

目的:探讨膜片钳实验中大鼠心肌细胞分离过程中影响细胞活性和钙耐受性的各种影响因素.方法:采用Langendorff灌流,先用台氏液灌流30s,再用无钙台氏液灌流5～7 min,其后换含胶原酶Ⅰ 0.1～0.2 g/L的低钙酶液消化约8～30min,最后用无钙台氏液冲洗心脏2 min,剪下心肌组织,KB液中37℃剪碎吹打温育5 min后,用200μm筛网过滤,室温静置换液后进行膜片钳实验.结果:注意控制水,酶,温度等各种因素后,可得到杆状、横纹清晰、膜良好的耐钙心肌细胞.结论:大鼠心肌细胞分离过程中各种因素控制好后可以保证一定的细胞成活率及其质量,有利于膜片钳实验的顺利进行.
목적:탐토막편겸실험중대서심기세포분리과정중영향세포활성화개내수성적각충영향인소.방법:채용Langendorff관류,선용태씨액관류30s,재용무개태씨액관류5～7 min,기후환함효원매Ⅰ 0.1～0.2 g/L적저개매액소화약8～30min,최후용무개태씨액충세심장2 min,전하심기조직,KB액중37℃전쇄취타온육5 min후,용200μm사망과려,실온정치환액후진행막편겸실험.결과:주의공제수,매,온도등각충인소후,가득도간상、횡문청석、막량호적내개심기세포.결론:대서심기세포분리과정중각충인소공제호후가이보증일정적세포성활솔급기질량,유리우막편겸실험적순리진행.
Objective: To investigate the factors affecting the viability and Ca2+ tolerance of isolated rats' cardiac myocytes for patch clamp research. Methods: Hearts were firstly perfused by the Langendorff perfusion apparatus with normal Tyrode' s solution, then with Ca2+-free Tyrode's solution and subsequently with Iow Ca2+ enzyme solution containing collagenase 0.1-0.2 g/L. All the solutions were saturated with oxygen and the perfusion temperature was kept at 37 ℃. Finally hearts were washed by Ca2+-free Tyrode' s solution, after which the ventricles were minced into small pieces in KB solution, dispersed and filtered. The isolated myocytes were stored in KB solution at room temperature for 1 h and recovered to normal calcium concentration before patch clamp experiments. Results: When all the factors such as water, enzyme, Ca2+ ,pH, and oxygen were well controlled, the well constructed and rod-like cardiac myocytes with a yielding rate of 30%-50% came out. Conclusion: All the factors should be well controlled, which ensured the isolated cells Ca2+ tolerant and appropriate for patch clamp experiments.