CAJ | 학술논문

目的探讨内质网应激在白蛋白超负荷诱导肾小管上皮细胞凋亡中的作用和分子机制.方法 Western印迹法检测肾小管上皮细胞(HKC)内质网伴侣蛋白糖调节蛋白78(GRP78)和内质网应激蛋白CHOP(CCAAT增强子结合蛋白同源蛋白,也称为GADD153)表达与人血清白蛋白(HSA)作用时间和浓度的关系.实时荧光定量PCR法检测CHOP siRNA对CHOP基因转录的抑制情况.Western印迹法检测CHOP siRNA转染后CHOP蛋白水平的变化.膜联蛋白V和碘化丙锭(Annexin V-FITC和PI)双染的流式细胞术检测白蛋白诱导HKC凋亡的变化以及CHOP siRNA对HKC凋亡的影响.结果 (1)分别以0、5、10、20 g/L白蛋白作用于HKC 24 h,GRP78、CHOP蛋白表达及细胞凋亡均随白蛋白浓度的增加而上调,各组间差异有统计学意义(P<0.01);以20 g/L白蛋白分别作用于HKC 0、6、12、24、36 h,GRP78蛋白表达在6 h即显著增加,CHOP蛋白表达及HKC凋亡则在12 h显著增加,各组间差异有统计学意义(P<0.01).(2)CHOP siRNA显著抑制白蛋白诱导的CHOP基因转录及蛋白表达(P<0.01),对白蛋白诱导的HKC凋亡有显著抑制作用(P<0.01).结论白蛋白超负荷可诱导肾小管上皮细胞发生内质网应激,并通过上调促凋亡因子CHOP表达引起肾小管上皮细胞内质网相关的细胞凋亡,这可能是蛋白尿引起肾小管间质病变的重要机制.
목적 탐토내질망응격재백단백초부하유도신소관상피세포조망중적작용화분자궤제.방법 Western인적법검측신소관상피세포(HKC)내질망반려단백당조절단백78(GRP78)화내질망응격단백CHOP(CCAAT증강자결합단백동원단백,야칭위GADD153)표체여인혈청백단백(HSA)작용시간화농도적관계.실시형광정량PCR법검측CHOP siRNA대CHOP기인전록적억제정황.Western인적법검측CHOP siRNA전염후CHOP단백수평적변화.막련단백V화전화병정(Annexin V-FITC화PI)쌍염적류식세포술검측백단백유도HKC조망적변화이급CHOP siRNA대HKC조망적영향.결과 (1)분별이0、5、10、20 g/L백단백작용우HKC 24 h,GRP78、CHOP단백표체급세포조망균수백단백농도적증가이상조,각조간차이유통계학의의(P<0.01);이20 g/L백단백분별작용우HKC 0、6、12、24、36 h,GRP78단백표체재6 h즉현저증가,CHOP단백표체급HKC조망칙재12 h현저증가,각조간차이유통계학의의(P<0.01).(2)CHOP siRNA현저억제백단백유도적CHOP기인전록급단백표체(P<0.01),대백단백유도적HKC조망유현저억제작용(P<0.01).결론 백단백초부하가유도신소관상피세포발생내질망응격,병통과상조촉조망인자CHOP표체인기신소관상피세포내질망상관적세포조망,저가능시단백뇨인기신소관간질병변적중요궤제.
Objective To investigate the effects and molecular mechanism of endoplasmic reticalam stress (ERS) on albumin-induced apoptosis in renal proximal tubular cells (HKCs). Methods Western blot was performed to detect the relationship of the expression of glucose-regulated protein 78 (GRF78) and CCAAT/enhancer-binding protein-homologous protein (CHOP) with the action time and concentration of haman serum albumin (HSA). Expression levels of CHPO mRNA and protein in HKCs after CHOP siRNA transfection were examined by real-time fluorescence quantitative PCR and Western blot respectively. Annexin-V-FITC and PI doable staining cytometry was used to detect the apoptosis of HKCs induced by HSA and influenced by CHOP siRNA. Results (1)After HKCs were stimulatde by 0, 5, 10, 20 g/L albumin for 24 hours respectively, the expression of GRP78, CHOP and HKCs apoptosis were increased with the albumin concentration (P<0.01). After HKCs were stimulated by 20 g/L albumin for 0, 6, 12, 24, 36 hours respectively, the expression of GRP78 was up-regulated at 6-hour, while CHOP and HKCs apoptosis were increased at 12-hour, and significant differences were found among groups (P<0.01). (2) CHOP siRNA significantly inhibited albumin-induced HKC CHOP mRNA and protein expression, as well as HKC apoptosis (P<0.01). Conclusions Renal tubular cells exposed to high protein load result in EBS. ERS may subsequently lead to tubular damage by activation of pro-apoptosis factor CHOP.