新生儿窒息后血清对人肾近曲小管上皮细胞线粒体膜通透性的影响*

赵婧1董文斌1李鹏云3王明勇2邓存良2许开桂1

(四川医科大学附属医院 1.新生儿科,2.传染与免疫研究室, 3.四川医科大学心肌电生理学研究室, 四川 泸州 64600)

【摘要】目的研究新生儿窒息后血清对人肾近曲小管上皮细胞(HK-2)线粒体膜通透性的影响。方法以人肾近曲小管上皮细胞(HK-2)为研究对象，实验共分为: 正常对照组、窒息组和吡咯烷二硫氨基甲酸(PDTC)干预组。以20%窒息后24小时血清作为攻击浓度。用四氯四乙基苯并咪唑基羰花青碘化物(JC-1)检测HK-2细胞线粒体膜电位变化；Western blot测HK-2细胞内线粒体中CytC的释放情况。结果①经窒息血清攻击后HK-2细胞线粒体膜红/绿荧光强度比值(590/530nm)明显降低(P<0.01)，但有了PDTC干预，HK-2细胞线粒体膜红/绿荧光强度比值明显升高(P<0.01)。②正常组HK-2细胞的胞浆中不含细胞色素C，主要存在于线粒体内，窒息组胞浆中细胞色素C从线粒体漏出较对照组明显增加，相应的在线粒体内表达减少，差异有统计学意义(P<0.05)；PDTC干预组胞浆中细胞色素C表达也有所增加，但大部分仍位于线粒体内，与窒息组相比，差异有统计学意义(P<0.05)。结论新生儿窒息后血清可影响人HK-2细胞线粒体膜通透性，使线粒体膜电位下降，膜间隙蛋白如细胞色素C等漏出。其胞内信号机制涉及到NF-κB活化。

【关键词】婴儿； 新生； 窒息； 凋亡； 线粒体； 核因子-κB；肾损伤

【中图分类号】R 722.12【文献标志码】A

基金项目：四川省杰出青年学科带头人培养基金(04ZQ026-033);四川省科技厅应用基础项目(2008JY0015)

通讯作者：董文斌，教授，E-mail: dongwenbin2000@163.com

收稿日期：( 2015-01-19； 编辑： 张文秀)

Effect of postasphyxial-serum in neonate on the mitochondria membrane permeability in renal tubular cellsZHAO Jing1,DONG Wenbin1,LI Pengyun2,etal

(1.DepartmentofNewbornMedicine，Luzhou646000,Sichuan,China; 2.LaboratoryofInfectionandImmunity,

TheAffiliatedHospitalofLuzhouMedicalCollege，Luzhou646000,Sichuan,China; 3.InstituteofMyocardium

Electrophysiology,SichuanMedicalUniversity,Luzhou646000,Sichuan,China)

Abstract【】ObjectiveTo investigate the effect of postasphyxial-serum in neonate on the mitochondria membrane permeability in renal tubular cells. MethodsHuman renal proximal tubular cell line HK-2 cell was used as target cell, which was divided into control group, asphyxia group and pyrrolodine dithiocarbamate (PDTC) blocking group. The attacking concentration of serum was 20%. The mitochondria membrane potential was detected by 5,5,6,6 -tetrachloro-1,1,3,3 -tetraethyl benzimidazolyl carbocyanine iodide(JC-1), and the release of the apoptogenic mitochondrial proteins cytochrome C was assessed by Western Blot.ResultsThe red/green fluorescence light intensity ratio of mitochondria membrane potential(590/530nm) in asphyxia group and PDTC blocking group were significantly lower than that in normal control group(P<0.01). The red/green fluorescence light intensity ratio of mitochondria membrane potential(590/530nm)in PDTC blocking group was significantly increased(P<0.01) compared with that of other groups. Cytochrome C was not present in the cytosolic fraction of normal cells. Increasing amounts of cytochrome C were detected in the cytosol of postasphyxial-serum treated cells, with a concomitant decrease of cytochrome C in the mitochondrial fraction(P<0.05). In contrast, no significant release of cytochrome C into the cytosol or change in the amount of cytochrome C in the mitochondrial fraction was detected in PDTC-treated cells(P<0.05).ConclusionThese data demonstrates that postasphyxial-serum is a stimulus, which can decrease the mitochondria membrane potential in human renal tubular cells and along with the release of cytochrome C from mitochondria to cytosol. It's intracellular signal transduction mechanism is presumably involved in the activation of nuclear factor-κB (NF-κB).

【Key words】Infant, Neonate; Asphyxia; Apoptosis; Mitochondria; Nuclear factor-κB; Renal injury

新生儿窒息可以导致多器官功能的损伤，肾损伤的发病率最高，其中肾小管又是肾损伤发生最早的部位[1]，而凋亡作为其损伤的一种形式已得到证实[2,3]。线粒体作为真核细胞内的"能量工厂"，其状态决定细胞死亡的类型。不同凋亡途径都是通过作用于线粒体膜来决定凋亡是否发生。线粒体膜电位的下降被认为是凋亡的早期事件[4]。线粒体膜通透性发生改变后还可能使膜间隙中的凋亡蛋白释放，细胞进入不可逆凋亡过程。……