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目的研究不同活性的胶原酶对大鼠心肌细胞存活率的影响。方法用4种不同活性的胶原酶以常规酶解法分离成年大鼠心肌细胞,观察分离即刻(D时点)、复钙60min后(E时点)和电刺激10min后(F时点)心肌细胞的存活率,采用IonOptix单细胞动缘检测系统同步检测心肌细胞收缩幅度(ph)、ph占单个心肌细胞长度(bl)的百分比(以ph/bl表示)、心肌细胞收缩速率(+dL/dt)和心肌细胞舒张速率(-dL/dt)等指标。结果观察D、E、F时点心肌细胞存活率,结果表明299U/mg胶原酶的分离效果286U/mg、203U/mg和403U/mg的胶原酶(P<0.05)。D时点4组心肌细胞的存活率没有差别,E、F时点时存活率降低。在心肌细胞收缩/舒张功能的检测中,299U/mg的胶原酶分离效果最佳,203、286、299U/mg和403U/mg组的ph分别是0.13±0.05、0.14±0.06、0.14±0.06μm和0.11±0.05μm;ph/bl分别是8.8%±2.4%、8.9%±2.3%、9.2%±3.5%和7.5%±2.2%;+dL/dt分别是1.8±0.6、1.9±0.6、2.0±0.7μm/s和1.7±0.5μm/s,-dL/dt分别是1.9±0.6、2.0±0.7、2.1±0.6μm/s和1.7±0.6μm/s。结论采用一定范围(203~299U/mg)的胶原酶对心肌细胞进行分离,酶活性越大,消化时间越短,心肌细胞存活率高、收缩/舒张功能好,其中以299U/mg活性的胶原酶效果最好。活性偏高或偏低的胶原酶分离心肌细胞的存活率和收缩/舒张功能均明显降低。
Objective To study the effects of collagenase with different activities on the survival rate of rat cardiomyocytes. Methods Cardiomyocytes were isolated from adult rat cardiomyocytes by conventional enzymatic hydrolysis using four collagenases with different activities. The myocardial cells were isolated immediately (D time point), 60 min after calcium supplementation (E time point) and 10 min after electrical stimulation (F time point) (Ph), the percentage of ph in single cardiomyocyte length (bl) (in ph / bl), the rate of cardiomyocyte contraction (+ dL / dt ) And cardiomyocyte relaxation rate (-dL / dt) and other indicators. Results The survival rate of cardiomyocytes at D, E and F was observed. The results showed that the separation efficiency of 299U / mg collagenase was 286U / mg, 203U / mg and 403U / mg collagenase (P <0.05). There was no difference in the viability of cardiomyocytes between D group and D group, and the survival rate decreased at E, F time points. In the detection of systolic / diastolic function of cardiomyocytes, the optimal isolation of collagenase was 299U / mg, and the phs of 203, 286, 299U / mg and 403U / mg were 0.13 ± 0.05, 0.14 ± 0.06 and 0.14 ± 0.06μm And 0.11 ± 0.05 μm respectively; ph / bl was 8.8% ± 2.4%, 8.9% ± 2.3%, 9.2% ± 3.5% and 7.5% ± 2.2%, respectively; dL / dt was 1.8 ± 0.6,1.9 ± 0.6,2.0 ± 0.7 μm / s and 1.7 ± 0.5 μm / s, and the -dL / dt was 1.9 ± 0.6, 2.0 ± 0.7, 2.1 ± 0.6 μm / s and 1.7 ± 0.6 μm / s, respectively. CONCLUSION: Myocardial cells are isolated by a certain range of collagenases (203 ~ 299U / mg). The greater the enzyme activity, the shorter the digestion time, the higher the survival rate of myocardial cells and the better the systolic / diastolic function. Among them, 299U / mg collagen Enzyme best. High or low activity of collagenase cardiomyocytes isolated from the survival rate and systolic / diastolic function were significantly lower.