缺氧-复氧对培养的大鼠心肌细胞损伤与细胞凋亡的影响

目的:探讨体外培养的大鼠心肌细胞在缺氧(缺血)和缺氧-复氧(再灌注)状态下心肌细胞损伤和细胞凋亡情况。方法:常规方式培养SD大鼠心肌细胞。给予模拟缺血(缺氧)溶液、模拟复氧(再灌注)液以及终浓度为200U/mL的超氧化无歧化酶(SOD)干预处理,制备缺氧-复氧损伤组(H/R组)、SOD+缺氧-复氧损伤组(SOD+H/R组)和缺氧预处理组(HP组)模型,未经处理的为正常对照组(control组)。结果:屹H/R组的细胞存活率较对照组明显降低(P<0.01);H/R+SOD组和HP组的细胞存活率虽然较正常对照组明显降低(P<0.05),但比H/R组为高(P<0.05);亿H/R组培养液中培养液中心肌酶活性显著高于HP组和H/R+SOD组(P<0.01)。役H/R组心肌细胞内的脂质过氧化产物丙二醛(MDA)含量也明显增高,与HP组和H/R+SOD组的心肌细胞内MDA含量比较有显著性差异(P<0.05);而HP组、H/R组与对照组间比较,无明显差异(P>0.05);均提示了缺氧-复氧过程中心肌细胞损伤严重,也说明了缺氧(血)预处理或SOD均有细胞保护作用。臆H/R组心肌细胞内游离钙含量增加,显著高于HP组和H/R+SOD组(P<0.01);逸流式细胞仪测定出H/R组心肌细胞凋亡率也明显高于HP组和H/R+SOD组(P<0.01)。心肌细胞凋亡的数量与细胞内钙的增加呈正相关(P<0.01)。肄电镜下所见H/R组的许多细胞表现为胞浆减少,核深染、固缩状态,异染色质不均匀,部分线粒体呈空泡变性,或是肌浆网囊泡变等凋亡期或凋亡前期样改变,少数呈凋亡向坏死方面改变;而其他组则少见此些变化。结论:体外培养的心肌细胞在缺氧-复氧情况下同样可加重心肌细胞损伤,可能是通过降低细胞内钙离子浓度而起作用的。

Effect of hypoxia-reoxygenation on cell injury and apoptosis in cultured rat myocytes

Objective: To study the effects of hypoxia-reoxygenation on cell injury and apoptosis in cultured neonatal rat myocytes. Methods: Cultured myocytes of neonatal SD rats have been divited into four groups-Hypoxia/Reoxygenation(Reperfusion)(H/R), Hypoxia/Reperfusion+SOD (H/R+SOD), Hypoxia Preconditioning(HP) and Control. Results: Myocyte viability in H/R group was much lower than that in contro, H/R+SOD and HP groups(P<0.05). The cultural contents of LDH, the myocyte contents of MDA and [Ca2+]i were also higher than those in any other groups(P<0.05). The myocyte apoptotic rate in H/R was also higher (18.52±6.15)% and positively correlated with the increase of [Ca2+]i level in each group(γ=0.92), but a significant lower incidence of apoptosis in HP and H/R+SOD groups compared with H/R (4.62±2.57)% and (6.94±2.71)%,respectively,P<0.01). Most cells in H/R group likely represent an early stage of apoptosis, such as compaction of nuclear chromatin into sharply circumscribed, uniformly dense masses that abut the nuclear envelope. Some were in advanced stage of apoptosis, such as shrinkage of the cytoplasm, variable density chromatin condensation, diffuse distribution. Conclusion: Hypoxia/reoxygenation was not only able to increase myocardiac injury, but also trigger and accelerate myocyte apoptosis. The overload of intracellular Calcium may be one of the causes of myocyte apoptosis.