高糖对人肾小管上皮细胞氧化应激及蛋白质损伤效应的实验研究

Effects of oxidative stress and its secondary protein impairment induced by high glucose content in human renal tubular epithelial cell

目的探讨高糖对人肾小管上皮细胞(HKC)氧化应激及蛋白质氧化损伤的作用。方法将体外培养的HKC分为3组:对照组、高糖短期处理组、高糖长期处理组。对照组葡萄糖浓度为5.5mmol/L,高糖处理组葡萄糖浓度为30mmol/L。高糖短期处理组分为6个时间点,即30min及1、6、12、24、48h高糖刺激组;高糖长期处理组为高糖连续刺激2个月。用活性氧(ROS)捕获剂氯甲基二氯二氢荧光素二乙酯(CM-H2DCFDA)孵育各组细胞,通过流式细胞技术和激光共聚焦显微镜检测高糖处理后不同时间点HKC中ROS的荧光强度以反映细胞内ROS的水平,用共聚焦显微镜检测荧光探针JC-1的荧光强度来表示线粒体膜电位,用2,4-二硝基苯肼(DNPH)比色法测定细胞内蛋白质羰基含量的变化。结果流式细胞技术和激光共聚焦显微镜检测显示,经高糖刺激后,HKC内ROS含量在1h时达高峰,随后逐渐降低;线粒体膜电位在高糖刺激后短时间内(1h)有增高,其后下降;DNPH比色法显示,短时高糖刺激对细胞中蛋白质羰基含量影响不明显(P>0.05),高糖刺激2个月时蛋白质羰基含量与短时刺激相比明显升高(P<0.05)。结论高糖处理可使HKC细胞氧化应激增强,从而造成生物大分子蛋白质的损伤。

Objective To explore the effects of oxidative stress and its secondary protein impairment induced by high glucose content in human kidney proximal tubular epithelial cell line （HKC）. Methods Cultured HKC were used to detect the reactive oxygen species （ROS） and protein carbonyl content. HKC were divided into three different groups： control group （normal glucose）, short-term treatment with high glucose group and long-term treatment with high glucose group. The glucose concentrations in control group and the high glucose treatment groups were 5.5mmol/L and 30mmol/L, respectively. Six time-points were set in short-term treatment with high glucose group namely 30min, 1h, 6h, 12h, 24h and 48h; glucose treatment was instituted for 2 months in long-term treatment of high glucose group. 5-（and-6）-chloromethyl-2′,7′-dichlorodihydrolquorescein diacetate, acetyl ester （CM-H2DCFDA） was used as ROS trapping agent, and flow cytometry and laser confocal microscopy were used to detect the concentration of ROS in HKC after treatment with high glucose for different periods of time. Flourescent probe JC-1 was used to test the change in mitochondrion membrane potential （MMP）. The protein carbonyl content was determined by 2,4-dinitrobenzene hydralazine （DNPH） chromatometry. Results The ROS level in HKC reached the peak at 1 hour after treatment with high glucose, and it then decreased gradually （P〈0.05）. The fluorescent intensity of JC-1 in- creased at 1 hour after the beginning of high glucose stimulation, and then decreased. No significant change occurred in protein carbonyl content within 48h treatment with high glucose （P〉0.05）, but after 2 months, there was significant change in the content of DNPH （P〈0.05）. Conclusion High glucose-induced oxidative stress in HKC can significantly elevate the protein carbonyl content.