缺氧与氧化损伤后肾小管上皮细胞能量代谢及细胞骨架的改变

Changes in energy metabolism and cytoskeleton in renal tubular epithelial cells after anoxia and oxidative stress

目的:观察缺氧、氧化损伤后培养的人肾小管上皮细胞能量代谢及细胞骨架改变之间的关系。方法:实验于2002-09/2003-09在解放军第三军医大学西南医院实验动物中心完成。①将人肾小管上皮细胞株HK-2细胞分为正常组、缺氧组、氧化损伤组3组:正常组中加入无血清RMPI1640培养液;缺氧组加入含0.1μmol/L抗霉素A的无血清无底物RMPI1640培养液;氧化损伤组加入含0.1mmol/LH2O2的无血清RMPI1640培养液。②观察各组细胞生长状况和形态及细胞存活率;测定细胞外液乳酸脱氢酶比活性代表肾小管上皮细胞膜通透性变化;采用ATP酶(Na+,K+和Ca2+-ATP)试剂盒直接测定各组细胞能量代谢改变,采用细胞免疫荧光化学方法检测各组细胞细胞骨架改变。结果:①正常组细胞生长状况良好,折光性强;缺氧、氧化损伤组细胞皱缩、突起减少,细胞存活率明显低于正常组[(56.2±2.3)%,(62.5±2.6)%,(96.5±3.2)%,P<0.01]。②缺氧、氧化损伤组乳酸脱氢酶比活性显著高于正常组(P<0.01)。③Na+-K+-ATP酶和Ca2+-ATP酶活性正常组明显高于缺氧、氧化损伤组[(9.61±0.53),(3.68±0.27),(5.10±0.19)nkat/g;(7.97±0.66),(3.12±0.24),(4.56±0.32)nkat/g,P<0.01],而缺氧组、氧化损伤组之间比较差异不显著(P>0.05)。④免疫荧光标记细胞内细胞骨架(微管、微丝、中间丝)显示正常TEC微管、微丝、中间丝表达丰富,在胞浆内呈均匀一致分布;缺氧、氧化损伤后细胞骨架破坏、塌陷,网络结构和支架作用消失。结论:缺氧、氧化损伤能量代谢障碍可造成细胞骨架破坏,最终导致肾小管上皮细胞的坏死和凋亡。

AIM： To study the relationship between energy metabolism and changes of eytoskeleton in cultured renal tubular epithelial cells （TEC） after anoxia and oxidative stress. METHODS： The experiment was accomplished in the Experimental Animal Center of Southwest Hospital of Third Military Medical University of Chinese PLA between September 2002 and September 2003. ①Human renal TEC cell strain HK-2 were divided into 3 groups. Normal control group： RMP11640 serum-free medium was added. Anoxia group： 0.1μnol/L antimycin A in RMPI1640 free-substrate and free-serum medium was added. Oxidative group： 0.1 mmol/L H202 in RMPI1640 free-serum medium was added.②The growth, form and survival rate of the cells were observed respectively, and changes of permeability in TEC cell membrane was detected by measuring the activity of lactate dehydrogenase （LDH） in extracellular fluid. ATPase kit （Na^＋, K^＋ and Ca^2＋-ATP） was adopted to detect the energy metabolism changes of cells in each group, and immunofluorescence was used to detect the cytoskeleton changes in TEC. RESULTS： ①The growing status and reflected light nature of TEC were better in the normal control group, while the shrinkage and enations were decreased, in anoxia and oxidative stress group, and the survival rate of cells were obviously lower than the normal control group [（56.2±2.3）% （62.5±2.6）%, （96.5±3.2）%, P 〈 0.01]. ②The activity of LDH were significantly higher in the anoxia group and oxidative stress group than the normal control group （P 〈 0.01）. ③The activities of Na^＋-K^＋-ATPase, Ca^2＋-ATPase in the normal control group was remarkably higher than anoxia group and oxidative stress group [（9.61 ±0.53）, （3.68±0.27）, （5.10 ±0.19）nkat/g; （7.97 ±0.66）, （3.12±0.24）, （4.56 ±0.32）nkat/g,P 〈 0.01], while there were no marked differences between the anoxia group and oxidative stress group. ④ The immunofluorescence labeled skeleton （microbubule, microfilamnet, intermediate filament） manifested that the expressions of normal TEC microbubule, microfilamnet and intermediate filament were plentiful, which were evenly distributed in the plasm. While the cell skeleton were destroyed and sank with network structure and skeleton disappeared in the anoxia group and oxidative stress group. CONCLUSION： The skeleton changes and energy metabolism disorder induced by anoxia and oxidative stress can cause the necrosis and apoptosis of TEC.