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  

Effect of postasphyxial-serum in neonate on the mitochondria membrane permeability in renal tubular cells

作　　者：赵婧 董文斌 李鹏云 王明勇 邓存良 许开桂 

机构地区：[1]四川医科大学附属医院新生儿科,四川泸州64600 [2]四川医科大学附属医院 [3]四川医科大学心肌电生理学研究室,四川泸州64600 [4]四川医科大学附属医院传染与免疫研究室,四川泸州64600

出　　处：《西部医学》2015年第5期652-655,659,共5页Medical Journal of West China

基　　金：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)

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。Objective To investigate the effect of postasphyxial-serum in neonate on the mitochondria membrane permeability in renal tubular cells. Methods Human 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 at- tacking 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. Results The red/green fluorescence light intensity ratio of mitochondria mem- brane 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 ceils. Increasing amounts of cytochrome C were detected in the cyto- sol of postasphyxial-serum treated cells, with a concomitant decrease of cytoehrome 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）. Conclusion These data demonstrates that postas- phyxial-serum is a stimulus, which can decrease the mitochondria membrane potential in human renal tubular cells and a- long 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）.

关 键 词：婴儿 新生 窒息 凋亡 线粒体 肾损伤 核因子-ΚB 

分 类 号：R722.12[医药卫生—儿科]