CAJ | 학술논문

目的观察造影剂对肾小管上皮细胞自噬及凋亡的影响,探讨自噬在造影剂诱导的肾小管上皮细胞损伤中的作用.方法体外培养大鼠肾小管上皮细胞(NRK-52E),以不同浓度(25、50、100、200 gI/L)造影剂碘普罗胺处理NRK-52E细胞1h或以50 gI/L碘普罗胺刺激NRK-52E细胞不同时间(0.5、1、3、6、12、24 h),吖啶橙染色观察细胞内自噬体形成,Western印迹检测自噬标志蛋白微管相关蛋白1轻链3(LC3)的表达,流式细胞术检测细胞凋亡率.雷帕霉素(Rap,1μg/L)或3-甲基腺嘌呤(3-MA,2 mmol/L)与碘普罗胺(50 gI/L)共处理肾小管上皮细胞后,Western印迹法检测各组LC3及自噬相关蛋白Beclin-1的表达,绿色荧光蛋白标记的LC3(GFP-LC3)转染后荧光显微镜观察GFP-LC3的表达,Hoechst-33342染色观察细胞凋亡.结果 (1)随碘普罗胺刺激浓度增加肾小管上皮细胞自噬体表达先上升后下降.(2)随碘普罗胺刺激浓度及时间的增加肾小管上皮细胞LC3-Ⅱ/LC3-Ⅰ的表达先上升后下降(P＜0.05).(3)碘普罗胺刺激后,肾小管上皮细胞的凋亡率呈浓度及时间依赖性增加(P＜0.05).(4)Rap与碘普罗胺共处理肾小管上皮细胞后,LC3-Ⅱ/LC3-Ⅰ、Beclin-1及GFP-LC3表达上调,细胞凋亡减少(P＜0.05);3-MA与碘普罗胺共处理肾小管上皮细胞后,LC3-Ⅱ/LC3-Ⅰ、Beclin-1及GFP-LC3表达下调,细胞凋亡增加(P＜0.05).结论造影剂可诱导肾小管上皮细胞凋亡和自噬,小剂量造影剂刺激下肾小管上皮细胞自噬和凋亡同时发生,但高峰早于凋亡.在刺激早期一定程度增强自噬可抑制细胞凋亡,反之抑制自噬后细胞凋亡增加.
목적 관찰조영제대신소관상피세포자서급조망적영향,탐토자서재조영제유도적신소관상피세포손상중적작용.방법 체외배양대서신소관상피세포(NRK-52E),이불동농도(25、50、100、200 gI/L)조영제전보라알처리NRK-52E세포1h혹이50 gI/L전보라알자격NRK-52E세포불동시간(0.5、1、3、6、12、24 h),아정등염색관찰세포내자서체형성,Western인적검측자서표지단백미관상관단백1경련3(LC3)적표체,류식세포술검측세포조망솔.뢰파매소(Rap,1μg/L)혹3-갑기선표령(3-MA,2 mmol/L)여전보라알(50 gI/L)공처리신소관상피세포후,Western인적법검측각조LC3급자서상관단백Beclin-1적표체,록색형광단백표기적LC3(GFP-LC3)전염후형광현미경관찰GFP-LC3적표체,Hoechst-33342염색관찰세포조망.결과 (1)수전보라알자격농도증가신소관상피세포자서체표체선상승후하강.(2)수전보라알자격농도급시간적증가신소관상피세포LC3-Ⅱ/LC3-Ⅰ적표체선상승후하강(P＜0.05).(3)전보라알자격후,신소관상피세포적조망솔정농도급시간의뢰성증가(P＜0.05).(4)Rap여전보라알공처리신소관상피세포후,LC3-Ⅱ/LC3-Ⅰ、Beclin-1급GFP-LC3표체상조,세포조망감소(P＜0.05);3-MA여전보라알공처리신소관상피세포후,LC3-Ⅱ/LC3-Ⅰ、Beclin-1급GFP-LC3표체하조,세포조망증가(P＜0.05).결론 조영제가유도신소관상피세포조망화자서,소제량조영제자격하신소관상피세포자서화조망동시발생,단고봉조우조망.재자격조기일정정도증강자서가억제세포조망,반지억제자서후세포조망증가.
Objective To observe the effect of contrast media on autophagy and apoptosis of renal tubular epithelial cells,evaluate the role of autophagy in contrast media-induced renal tubular epithelial cells injury.Methods NRK-52E cells were exposed to iopromide at different concentration for 1 hour or at 50 gI/L for variable incubation time.Rapamycin (1 μg/L) and 3-methyadenine (2 mmol/L) were further introduced to investigate the role of autophagy in the process.The formation of autophagy was observed by acridine orange staining and Green fluorescent protein tagged LC3 (GFP-LC3).The expression of autophagy protein LC3 and Beclin-1 was examined by Western blotting,and the apoptosis level was examined by flow cytometry and Hoechst 33342-staining.Results (1) Autophagy could be enhanced by contrast media in renal tubular epithelial cells.(2) The expression of LC3-Ⅱ/LC3-Ⅰ in renal tubular epithelial cells rose at first and then dropped with the increase of iopromide stimulation time and concentration (P ＜ 0.05).(3) Iopromide promoted renal tubular epithelial cell apoptosis in dose-and time-dependent manner (P ＜ 0.05).(4) Co-culture with rapamycin further increased LC3-Ⅱ/LC3-Ⅰ,Beclin-1 and GFP-LC3 expression,but obviously prevented iopromide-induced apoptosis of renal tubular epithelial cells (P ＜ 0.05).On the contrary,Coculture with 3-methyadenine reduced iopromide-induced LC3-Ⅱ/LC3-Ⅰ,Beclin-1 and GFP-LC3 overexpression,but aggravated the apoptosis induced by iopromide (P＜0.05).Conclusions Contrast media can induce renal tubular epithelial cells apoptosis as well as autophagy.Enhancing autophagy appropriately has a protective effect on iopromide-induced renal tubular epithelial cells apoptosis,which conforms that autophagy plays an important role in antagonizing iopromide-induced renal tubular epithelial cells injury.