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目的:研究环维黄杨星D(CVB-D)对培养乳鼠心肌细胞缺氧/复氧损伤的保护作用和对急性分离大鼠心肌细胞内游离Ca2+浓度的影响。方法:采用培养的乳鼠心肌细胞建立缺氧/复氧损伤模型,测定心肌细胞搏动频率、细胞存活率、肌酸激酶(CK)的含量;应用特异性荧光指示剂Fluo-3/AM负载急性分离的大鼠心肌细胞,用激光共聚焦显微镜检测胞内游离钙的变化。结果:缺氧/复氧损伤+环维黄杨星D组(H/R+CVB-D)乳鼠心肌细胞搏动频率、细胞存活率与缺氧/复氧损伤组比较明显升高,CK含量与缺氧/复氧损伤组比较明显下降。对急性分离大鼠心肌细胞内[Ca2+]i逐步升高,并呈剂量依赖性;在有外钙和无外钙时,CVB-D对胞内钙的升高有显著差异(P<0.05);在无外钙并加入内罗啶孵育后,CVB-D引起的胞内[Ca2+]i轻度升高,但与无钙组相比无显著性差异(P>0.05)。结论:环维黄杨星D对培养乳鼠心肌细胞缺氧/复氧损伤有保护作用,对急性分离大鼠有升高心肌细胞内游离钙离子浓度的作用,[Ca2+]的升高既源于内钙释放又源于外钙内流。
OBJECTIVE: To study the protective effect of cyclovirobuxine D (CVB-D) on cultured neonatal cardiomyocytes against hypoxia/reoxygenation injury and its effect on the concentration of free Ca2+ in myocardial cells of acutely isolated rats. METHODS: Hypoxic/reoxygenation injury model was established in cultured neonatal rat cardiomyocytes. The beating frequency, cell survival rate and creatine kinase (CK) content were measured. The specific fluorescence indicator Fluo-3/AM was used to load acutely. Isolated rat cardiomyocytes were examined for changes in intracellular free calcium by laser confocal microscopy. RESULTS: Hypoxia/reoxygenation injury + cyclovirobuxine group D (H/R+CVB-D) beat frequency and cell survival rate of neonatal rat cardiomyocytes increased significantly compared with hypoxia/reoxygenation injury group. Hypoxia / reoxygenation injury group decreased significantly. The [Ca2+]i increased gradually in a dose-dependent manner in acutely isolated rat cardiomyocytes, and there was a significant difference in the increase of intracellular calcium in CVB-D with and without extracellular calcium (P<0.05). In the absence of external calcium and incubation with endorphin, CVB-D induced a slight increase in intracellular [Ca2+]i, but there was no significant difference compared with the non-calcium group (P>0.05). CONCLUSION: Cyclovirobuxine D has protective effects on cultured neonatal cardiomyocytes against hypoxia/reoxygenation injury, and it has an effect on the increase of intracellular free calcium ion concentration in acutely isolated rat myocardial cells. [Ca2+] rises from both Internal calcium release is also derived from extracellular calcium influx.