CAJ | 학술논문

目的探讨人类血清白蛋白(HSA)超负荷损伤肾小管上皮细胞后对肾间质微血管损伤的影响及可能机制.方法激光共聚焦显微镜观察肾小管上皮细胞(HKC)吞饮罗丹明标记白蛋白(TRITC-BSA)以及吞饮受体cubilin siRNA对其的抑制效应.氢化乙锭标记的荧光探针检测白蛋白刺激HKC产生O2-以及白蛋白吞饮受体cubilin siRNA和线粒体呼吸链复合物I抑制剂鱼藤酮对HKC产生O2-的抑制作用.培养上清H2O2采用化学比色方法检测.倒置显微镜下观察HSA激活的HKC以及cubilin siRNA或鱼藤酮预处理的HKC与脐静脉内皮细胞(HUVEC)共培养后血管内皮管样结构形成情况.四甲基偶氮唑盐(MTT)比色法测定内皮细胞活力.用流式细胞仪以AnnexinV FTTC/PI双染法检测内皮细胞凋亡率.结果 (1)cubilin siRNA可显著抑制HKC吞饮白蛋白(P＜0.05).(2)HSA刺激HKC产生ROS呈剂量及时间依赖性(P＜0.05);cubilin siRNA及鱼藤酮均可抑制白蛋白超负荷诱导HKC产生大量ROS(P＜0.05).(3)HKC与HUVEC共培养体系中,HSA活化的HKC抑制内皮细胞管样结构的形成,内皮细胞增殖显著减少(P＜0.05),细胞凋亡率显著增高(P＜0.05);而cubilin siRNA和鱼藤酮干预组内皮细胞管样结构数及内皮细胞活力显著增加(P＜0.05),细胞凋亡率显著降低(P＜0.05).结论白蛋白可通过吞饮受体cubilin激活肾小管上皮细胞线粒体呼吸链复合物I产生大量ROS,后者可能是慢性肾病时大量蛋白尿活化肾小管上皮细胞进而损伤肾小管周微血管网的重要介质.
목적 탐토인류혈청백단백(HSA)초부하손상신소관상피세포후대신간질미혈관손상적영향급가능궤제.방법 격광공취초현미경관찰신소관상피세포(HKC)탄음라단명표기백단백(TRITC-BSA)이급탄음수체cubilin siRNA대기적억제효응.경화을정표기적형광탐침검측백단백자격HKC산생O2-이급백단백탄음수체cubilin siRNA화선립체호흡련복합물I억제제어등동대HKC산생O2-적억제작용.배양상청H2O2채용화학비색방법검측.도치현미경하관찰HSA격활적HKC이급cubilin siRNA혹어등동예처리적HKC여제정맥내피세포(HUVEC)공배양후혈관내피관양결구형성정황.사갑기우담서염(MTT)비색법측정내피세포활력.용류식세포의이AnnexinV FTTC/PI쌍염법검측내피세포조망솔.결과 (1)cubilin siRNA가현저억제HKC탄음백단백(P＜0.05).(2)HSA자격HKC산생ROS정제량급시간의뢰성(P＜0.05);cubilin siRNA급어등동균가억제백단백초부하유도HKC산생대량ROS(P＜0.05).(3)HKC여HUVEC공배양체계중,HSA활화적HKC억제내피세포관양결구적형성,내피세포증식현저감소(P＜0.05),세포조망솔현저증고(P＜0.05);이cubilin siRNA화어등동간예조내피세포관양결구수급내피세포활력현저증가(P＜0.05),세포조망솔현저강저(P＜0.05).결론 백단백가통과탄음수체cubilin격활신소관상피세포선립체호흡련복합물I산생대량ROS,후자가능시만성신병시대량단백뇨활화신소관상피세포진이손상신소관주미혈관망적중요개질.
Objective To explore the influence of albumin-activated renal tubular epithelial cells (RTECs)on peritubular capillaries in co-culture system and its potential mechanism. Methods Endocytosis of TRITC labeled bovine scrum albumin (TRITC-BSA) by HKC was detected by laser scanning confocal fluorescence microscope. HKC or HKC transfected with cubilin (endocytic receptor of albumin) siRNA or pre-treated with rotenone was incubated with albumin(20 g/L) for 24 h respectively. Fluorescence probe technique and spectrometry were applied for determination of intracellular superoxide anion O2-and H2O2 in supematant. Then, the albumin-aetivated-HKC, pretreated-HKC with cubilin siRNA or rotenone, was cultured with HUVEC for 24 h in co-culture system respectively. HUVEC proliferation was determined by MTT and cellular apoptosis was analyzed by flow cytometry. Tabular morphogenesis of endothelial cells was examinedby microscopy. Results TRITC-BSA uptake was obviously lower in HKC transfected with cubilin siRNA. Intracellular generation of O2-and H2O2 in culture supernatant was increased in dose-and time-dependent manner after stimulating with albumin. The levels of O2-and H2O2 were suppressed by cubilin siRNA and rotenone. In co-culture system, albumin-activated-HKC induced endothelial cells apoptosis and inhibited their capillary tubular morphogenesis. Pretreatment of HKC with cubilin siRNA or rotenone could suppress endothelial cells apoptosis and promote capillary tubular morphogenesis. Conclusions There may be a crosstalk between RTECs and peritubular microvascular endothelial cells in renal proteinurie diseases. The generation of ROS by albumin-activated RTECs may play an important role in this process.