Ⅱ型胶原酶加压灌流提高成年大鼠心肌细胞分离效率李德，唐兵，杨大春.docVIP

PAGE PAGE 4 Ⅱ型胶原酶加压灌流提高成年大鼠心肌细胞分离效率 李德，唐兵，杨大春，杨永健 成都军区总医院心内科（成都610083） 摘要：目的 探讨效率更高的成熟心肌细胞分离方法。方法 采用Ⅱ型胶原酶升主动脉逆行灌流法分离成年大鼠心肌细胞。对照组采用Langendorff装置灌流；实验组在Langendorff装置基础上加压匀速灌流。在倒置显微镜下观察细胞形态，并计算杆状细胞比率及产量；通过台盼蓝染色和局部场剌激评价心肌细胞活性。结果 分离即刻，实验组杆状细胞比率显著高于对照组（90.3±4.4% νs 53.4±5.2%，p0.01）；实验组活细胞产量也显著高于对照组[（3.6±0.7）×107 νs（1.9±0.6）×107 ，p0.01]。复钙过程中，实验组发生自发性收缩的细胞比率及变圆细胞比率均明显低于对照组（10.4±2.1% νs 18.9±4.5%，p0.01；5.3±1.3%νs 8.6±1.7%，p0.05 ）。实验组台盼蓝染色阴性的杆状细胞比率和局部场剌激条件下收缩细胞比率明显高于对照组（95.3±8.2% νs 90.6±9.8%，p0.05；92.2±7.6% νs 85±6.4%，p0.01）。结论 Ⅱ型胶原酶加压灌流可获得高产量、高活性的心肌细胞，提高了成熟心肌细胞的分离效率。 关键词：大鼠；心肌细胞；细胞分离 Pressurizing perfusion with collagenase typeⅡ improve the isolation efficacy of adult rat cardiomyocytes LI De, TANG Bing, YANG Da-chun,YANG Yong-jian (Department of Cardiology, Chengdu Military General Hospital, Chengdu 610083, China.) Abstract: Objective To investigate a more effective method of isolation of adult rat cardiomyocytes. Methods Cardiac myocytes from SD rats were isolated by collagenase typeⅡperfusion in control group, and by pressurizing perfusion in experimental group. The cardiomyocytes were morphologically observed with optical microscope and evaluated by trypan blue dying and field stimulation. Results The ratio of rod-shaped cells and yield of viable cells per rat were significantly higher in experimental group than in control group[90.3±4.4% νs 53.4±5.2%，p0.01;（3.6±0.7）×107 νs（1.9±0.6）×107 ，p0.01]. During recalcification, The incidence of spontaneous contraction and deformation were significantly lower in experimental group than in control group（10.4±2.1% νs 18.9±4.5%，p0.01；5.3±1.3%νs 8.6±1.7%，p0.05 ）. The ratio of trypan blue dying negative cells and cells with contractile under field stimulation were significantly higher in experimental group than in control group also（95.3±8.2% νs 90.6±9.8%，p0.05；92.2±7.6% νs 85±6.4%，p0.01）. Conclusion Adult rat cardiomyocytes can be isolated more efficiently by collagenase typeⅡpressurizing perfusion. Key words: rat