rrBMP-7基因对缺氧复氧损伤心肌细胞的保护作用

Protective effects of transfecting bone morphogenetic protein-7 on rat cardiomyocytes against hypoxia-reoxygenation injury

目的探讨骨形态发生蛋白-7(BMP-7)基因对缺氧复氧损伤后心肌细胞的保护作用。方法将体外培养的乳大鼠心肌细胞分为3组:对照组(C组);缺氧复氧组(HR组):将培养的心肌细胞缺氧120min,复氧240min;转染组(BT组):将重组鼠BMP-7质粒转染心肌细胞后,再缺氧120min/复氧240min。每组重复8次。心肌细胞损伤程度以心肌细胞搏动功能评定、台盼蓝摄取率和乳酸脱氢酶(LDH)、磷酸肌酸激酶(CPK)活性来表示,同时检测心肌细胞超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量。通过荧光成像技术检测各组心肌细胞内钙含量的变化。结果与C组比较,HR组心肌细胞LDH、CPK含量显著增高,台盼蓝摄取率增加,SOD活性和细胞搏动频率显著下降,MDA和钙离子含量显著增高。与HR组比较,BT组心肌细胞台盼蓝摄取率降低,LDH活性减低,SOD活性增高,MDA含量下降,细胞内钙离子含量降低。结论BMP-7转染可对抗缺氧复氧对心肌细胞的损伤,其机制与提高心肌细胞抗氧化损伤能力,对抗缺氧复氧引起的细胞内钙超载有关。

Objective To investigate the protective effect of transfecting bone morphogenetic protein-7 （BMP-7） on cultured neonatal rat cardiomyocytes against hypoxia-reoxygenation injury. Methods Neonatal rat cardiomyocytes were cultured. The pcDNA rrBMP7 was introduced into cardiomyocytes by Fugene 6. 0 transfection method. The cardiomyocytes were divided into three groups ： control group （group C）, hypoxia-reoxygenation group （group HR） and gene transfecting group （group BT）. Trypan blue exclusion test was performed to detect cell viability. The activity of lactate dehydrogenase （LDH） and creatine phosphokinase （CPK） was assayed to evaluate cell injury. For evaluating the cell antioxidant ability, the activity of superoxide dismutase （SOD） and the level of malondialdehyde （MDA） were determined by colorimetric assay. Fluo-3 labeling method and confocal laser scanning microscopy were used to observe the change in intracellular calcium. Results The results showed that after 120 min of simulated ischemia followed by 240 min of reperfusion, cell pulsation rate was decreased, the activity of LDH, CPK and the trypan blue uptake rate were increased. As compared with the group C, SOD activity decreased and the content of MDA increased in Group HR. Compared with Group HR, the SOD activity increased and the content of MDA decreased in group BT. Treatment with BMP-7 gene transfecting led to a decrease of [Ca^2 ]i content in cardiomyocytes, showing that overloading of [Ca^2 ]i induced by hypoxia-reoxygenation was prevented （P〈0. 01）. Conelmion Transfecting BMP 7 gene could signifi cantly protect cardiomyocytes against hypoxia-reoxygenation injury. The mechanism may partly attribute to the suppression of [Ca^2 ]i overloading, and amelioration of oxidative damage in cardiomyocytes after hypoxia-reoxygenation.