国际泌尿系统杂志
國際泌尿繫統雜誌
국제비뇨계통잡지
INTERNATIONAL JOURNAL OF UROLOGY AND NEPHROLOGY
2012年
1期
20-25
,共6页
李兴%蒋玲琳%万辛%尹忠诚%曹长春
李興%蔣玲琳%萬辛%尹忠誠%曹長春
리흥%장령림%만신%윤충성%조장춘
肾小管%上皮细胞%维甲酸%大鼠
腎小管%上皮細胞%維甲痠%大鼠
신소관%상피세포%유갑산%대서
Kidney Tubules%Epithelial Cells%Tretinoin%Rats
目的 探讨全反式维甲酸(atRA)对大鼠肾小管上皮细胞缺氧性损伤的保护作用及其相关机制.方法 对大鼠肾小管上皮细胞进行体外培养,根据研究需要分四组,分别为对照组、CoCl2刺激组、atRA刺激组以及CoCl2与atRA共同刺激组,做如检测:用凝胶电泳迟滞分析(EMSA)测定各组别细胞NFκB活性,用qRT - PCR和Western blo分别检测各组别细胞NFκB亚单位p65,Scpep1、VEGF mRNA与蛋白表达水平,用免疫共沉淀检测NFκB亚单位p65与Scpep1相互作用.结果 CoCl2刺激组中VEGF mRNA与蛋白水平的表达明显高于对照组(P＜0.01).atRA刺激组中VEGF的mRNA与蛋白水平表达明显低于对照组(P ＜0.05).CoCl2刺激组中p65 mRNA与蛋白水平的表达明显高于对照(P＜0.01).atRA刺激组中VEGF 的mRNA与蛋白水平表达明显低于对照组(P＜0.05).共同刺激组中p65的表达受到抑制,与对照组比较,差异有统计学意义(P＜0.05).CoCl2刺激组中NFκB的DNA结合力明显高于正常组(P＜0.01).而atRA刺激组中NFκB的DNA结合力受到显著抑制,与对照组相比,差异有统计学意义(P＜0.05).在共同刺激组,NFκB的活性同样受到抑制,与对照组比,差异有统计学意义(P＜0.05).atRA刺激组中Scpep1mRNA与蛋白水平的表达水平显著高于对照组(P＜0.01).与对照组比较,CoCl2刺激组中Scpep1的mRNA与蛋白水平表达无明显变化,差异无统计学意义(P＞0.05).免疫沉淀结果显示,p65与Scpep1存在相互关联.结论 atRA通过Scpep1对NFκB复合物形成及其活性产生抑制效应,从而减弱VEGF的表达,对缺氧性损伤的肾小管上皮细胞发挥保护作用.
目的 探討全反式維甲痠(atRA)對大鼠腎小管上皮細胞缺氧性損傷的保護作用及其相關機製.方法 對大鼠腎小管上皮細胞進行體外培養,根據研究需要分四組,分彆為對照組、CoCl2刺激組、atRA刺激組以及CoCl2與atRA共同刺激組,做如檢測:用凝膠電泳遲滯分析(EMSA)測定各組彆細胞NFκB活性,用qRT - PCR和Western blo分彆檢測各組彆細胞NFκB亞單位p65,Scpep1、VEGF mRNA與蛋白錶達水平,用免疫共沉澱檢測NFκB亞單位p65與Scpep1相互作用.結果 CoCl2刺激組中VEGF mRNA與蛋白水平的錶達明顯高于對照組(P＜0.01).atRA刺激組中VEGF的mRNA與蛋白水平錶達明顯低于對照組(P ＜0.05).CoCl2刺激組中p65 mRNA與蛋白水平的錶達明顯高于對照(P＜0.01).atRA刺激組中VEGF 的mRNA與蛋白水平錶達明顯低于對照組(P＜0.05).共同刺激組中p65的錶達受到抑製,與對照組比較,差異有統計學意義(P＜0.05).CoCl2刺激組中NFκB的DNA結閤力明顯高于正常組(P＜0.01).而atRA刺激組中NFκB的DNA結閤力受到顯著抑製,與對照組相比,差異有統計學意義(P＜0.05).在共同刺激組,NFκB的活性同樣受到抑製,與對照組比,差異有統計學意義(P＜0.05).atRA刺激組中Scpep1mRNA與蛋白水平的錶達水平顯著高于對照組(P＜0.01).與對照組比較,CoCl2刺激組中Scpep1的mRNA與蛋白水平錶達無明顯變化,差異無統計學意義(P＞0.05).免疫沉澱結果顯示,p65與Scpep1存在相互關聯.結論 atRA通過Scpep1對NFκB複閤物形成及其活性產生抑製效應,從而減弱VEGF的錶達,對缺氧性損傷的腎小管上皮細胞髮揮保護作用.
목적 탐토전반식유갑산(atRA)대대서신소관상피세포결양성손상적보호작용급기상관궤제.방법 대대서신소관상피세포진행체외배양,근거연구수요분사조,분별위대조조、CoCl2자격조、atRA자격조이급CoCl2여atRA공동자격조,주여검측:용응효전영지체분석(EMSA)측정각조별세포NFκB활성,용qRT - PCR화Western blo분별검측각조별세포NFκB아단위p65,Scpep1、VEGF mRNA여단백표체수평,용면역공침정검측NFκB아단위p65여Scpep1상호작용.결과 CoCl2자격조중VEGF mRNA여단백수평적표체명현고우대조조(P＜0.01).atRA자격조중VEGF적mRNA여단백수평표체명현저우대조조(P ＜0.05).CoCl2자격조중p65 mRNA여단백수평적표체명현고우대조(P＜0.01).atRA자격조중VEGF 적mRNA여단백수평표체명현저우대조조(P＜0.05).공동자격조중p65적표체수도억제,여대조조비교,차이유통계학의의(P＜0.05).CoCl2자격조중NFκB적DNA결합력명현고우정상조(P＜0.01).이atRA자격조중NFκB적DNA결합력수도현저억제,여대조조상비,차이유통계학의의(P＜0.05).재공동자격조,NFκB적활성동양수도억제,여대조조비,차이유통계학의의(P＜0.05).atRA자격조중Scpep1mRNA여단백수평적표체수평현저고우대조조(P＜0.01).여대조조비교,CoCl2자격조중Scpep1적mRNA여단백수평표체무명현변화,차이무통계학의의(P＞0.05).면역침정결과현시,p65여Scpep1존재상호관련.결론 atRA통과Scpep1대NFκB복합물형성급기활성산생억제효응,종이감약VEGF적표체,대결양성손상적신소관상피세포발휘보호작용.
Objectives Background:It has been reported that the atRA - mediated protective effects in various cells are related to the inhibition of NFκB activities.There exists some evidence that an increase in VEGF,which is expressed by proximal tubular epithelial cells and regulated by NFκB,may play a critical role in maintaining peritubular capillary endothelium in renal disease.By stimulating the production of VEGF,hypoxia is involved in tubulointerstitial fibrosis processes in various renal diseases.Methods NRK52E cells survival rate was proportional to absorbance in dimethyl - thiazol - diphenyltetrazoliumbromide (MTT) tests.Quantitative Realtime PCR and Western blot were performed to assay the expression of VEGF、p65 and Scpep1.The activation of NFκB was determined by electrophoretic mobility shift assay ( EMSA ). Co - immunoprecipitation analysis demonstrates that whether the Scpep1 and NFκB protein interacted.Results We demonstrated that hypoxia - mimicking agent CoCl2 triggered hypoxia injury of rat proximal tubular epithelial cells and significantly reduced cell viability.In addition of atRA,we found that cell survival rate was increased.Under CoCl2 - mimicking hypoxia conditions,the expression of VEGF and p65,the main component of NFκB heterodimers increased and were significantly attenuated by addition of atRA.There was a similar variation of NFκB/DNA binding activities.atRA not only activated distinct pathways to stimulate the expression of Scpep1,a retinoid - inducible gene,under normoxic conditions but they also acted in CoCl2 - mimicking hypoxia.Conclusions The protective effects of atRA against hypoxia - induced injury might be involved in suppression of VEGF expression via stimulating Scpep1 distinct pathways and inhibiting NFκB pathway.
