大鼠心肌细胞分离方法的改进

An improved method of isolation of rat cardiac myocytes

目的介绍一种易于判断酶解终点的分离大鼠心肌细胞的方法。方法成年SD大鼠麻醉后开胸取出心脏,分别采用传统的Langendorff灌流装置和本实验室改进的Langen-dorff灌流装置,于恒温37℃灌流消化液(含0.6%胶原酶B,0.6%BSA,30μmol.L-1Ca2+的台氏液)分离细胞,并采用IonOptix单细胞动缘检测系统检测细胞功能。结果传统的Langendorff灌流装置分离细胞,酶解时间约13～16min,但此法终点难以判断,每次分离的细胞质量差别较大,且不耐钙,收缩的稳定性较差。而以改进的Langendorff灌流装置分离细胞易于确定消化终点,细胞成活率大于80%,在含1.8mmol.L-1Ca2+的台氏液中存活率也高达50%。给予电压15V、频率1Hz、波宽4ms的持续电刺激时,其在1000s内收缩舒张功能稳定。结论经改造的Langendorff灌流装置分离的心肌细胞成活率高,可控性强,结果稳定,能够更好地用于检测细胞收缩舒张功能的实验。

Aim To introduce an improved method of Langendorff perfusion of the isolated rat heart that is easy to determine the termination of digestion.Methods Hearts were excised quickly from anesthetized SD rats.After the perfusate was free of blood,the solution was changed to perfusion buffer（0.6% Collagenase B,0.6% BSA,30 μmol·L^-1 Ca2＋ in Tyrode solution）at 37℃.Hearts were isolated by traditional and improved Langendorff perfusion.Individual myocardial cell was measured by video-based motion edge-detection system（IonOptix,USA）.Results One group of heart was digested by traditional Langendorff perfusion for 13~16 min.The termination of digestion could not be judged properly by this method.The nature and quality of the cardiocytes were various.The cardiocytes could not keep their survival and viability when exposed to Tyrode Solution with 1.8 mmol·L^-1 Ca^2＋.Another group was digested by improved Langendorff perfusion.More than 80% survival cardiac ventricle myocytes could be obtained by improved Langendorff perfusion.Moreover,about 50% cardiac myocytes exposed to Tyrode solution with 1.8 mmol·L^-1 Ca^2＋ could retain rod-shaped and be used to contraction research.The contraction of cardiocyte was stabile within 1 000 s.Conclusion Cardiac myocytes disassociated from improved Langendorff perfusion can be used in these studies of detecting contraction and relaxation.This method is economical and easy to control.The beginners are able to acquire the technological method over a short-term practice.