CAJ | 학술논문

目的探讨微RNA?21(miR?21)对脯氨酸羟化酶2(proline hydroxylase domain 2, PHD2)的调控作用,及其对肾脏晚期缺血预适应(ischemic preconditioning,IPC)小鼠的保护作用. 方法建立肾脏晚期缺血预适应小鼠模型,分为两组:(1)缺血再灌注组(IR组):假手术后第4天双侧肾蒂夹闭35 min后恢复灌注;(2)缺血预适应组(IPC组):第1天双侧肾蒂夹闭15 min ,手术第4天处理同IR组.于术后第5天处死所有小鼠,留取血液及肾脏标本.HE染色观察肾组织病理变化;常规生化检测血肌酐(Scr)变化;实时荧光定量PCR法检测肾脏miR?21表达;Western印迹法检测肾组织PHD2和低氧诱导因子1α(HIF?1α)蛋白表达变化.体外给予人肾小管上皮细胞(HK?2)1% O2处理6 h ,采用Lipofectamine 2000法转染锁核酸(LNA)修饰的anti?miR?21寡核苷酸抑制miR?21表达,实时荧光定量PCR法检测细胞miR?21、PHD2以及HIF?1αmRNA表达;Western印迹法检测细胞PHD2与HIF?1α蛋白表达. 结果体内实验结果发现,与IR组相比,IPC组小鼠血肌酐和肾组织病理损伤有明显改善(均P<0.01);与IR组相比,IPC组小鼠肾组织miR?21表达上调(P<0.01);PHD2蛋白表达下调(P<0.05),HIF?1α蛋白表达上调(P<0.05).体外HK?2细胞低氧模型实验结果提示,抑制细胞miR?21表达可致PHD2蛋白水平明显升高(P<0.05). 结论 miR?21对PHD2具有负向调控作用.晚期IPC可能通过诱导miR?21表达,减少PHD2表达,间接上调HIF?1α,对肾脏缺血再灌注损伤起到保护作用.
목적 탐토미RNA?21(miR?21)대포안산간화매2(proline hydroxylase domain 2, PHD2)적조공작용,급기대신장만기결혈예괄응(ischemic preconditioning,IPC)소서적보호작용. 방법 건립신장만기결혈예괄응소서모형,분위량조:(1)결혈재관주조(IR조):가수술후제4천쌍측신체협폐35 min후회복관주;(2)결혈예괄응조(IPC조):제1천쌍측신체협폐15 min ,수술제4천처리동IR조.우술후제5천처사소유소서,류취혈액급신장표본.HE염색관찰신조직병리변화;상규생화검측혈기항(Scr)변화;실시형광정량PCR법검측신장miR?21표체;Western인적법검측신조직PHD2화저양유도인자1α(HIF?1α)단백표체변화.체외급여인신소관상피세포(HK?2)1% O2처리6 h ,채용Lipofectamine 2000법전염쇄핵산(LNA)수식적anti?miR?21과핵감산억제miR?21표체,실시형광정량PCR법검측세포miR?21、PHD2이급HIF?1αmRNA표체;Western인적법검측세포PHD2여HIF?1α단백표체. 결과 체내실험결과발현,여IR조상비,IPC조소서혈기항화신조직병리손상유명현개선(균P<0.01);여IR조상비,IPC조소서신조직miR?21표체상조(P<0.01);PHD2단백표체하조(P<0.05),HIF?1α단백표체상조(P<0.05).체외HK?2세포저양모형실험결과제시,억제세포miR?21표체가치PHD2단백수평명현승고(P<0.05). 결론 miR?21대PHD2구유부향조공작용.만기IPC가능통과유도miR?21표체,감소PHD2표체,간접상조HIF?1α,대신장결혈재관주손상기도보호작용.
Objective To investigate the molecular mechanism of protection of ischemia preconditioning on renal ischemia reperfusion injury. Methods Male C57/BL6N mice were randomly divided into two groups: in IR group, 35 min ischemia was induced by occlusion of both renal pedicles followed by 24 h perfusion (I/R). 15 min ischemia was induced 4 days before I/R in IPC group. Blood sample and kidney were collected in IR and IPC group after 24 h perfusion. Serum creatinine (Scr) and histological changes were used to evaluate the renal injury. PHD2 and HIF-1αwere evaluated by Western blotting, miR-21 expression was confirmed by real-time PCR. In vitro, hypoxic model was established by 1% O2 in HK-2 cells. Knockdown of miR-21 in hypoxic model was perfermed by locked nucleic acid modified-anti-miR-21 transfection. The levels of miR-21, HIF-1α and PHD2 mRNA were confirmed by real-time PCR. The levels of HIF-1α and PHD2 proteins were tested by Western blotting. Results In vivo, Compared with IR group, the renal function and histological changes were improved in IPC group (P<0.01). Compared with IR group, the expression of miR-21(P<0.01) and HIF-1α(P<0.05) were increased in IPC group, while PHD2 was reduced (P<0.01). In vitro, hypoxia reduced miR-21. The inhibition of miR-21 could increased the expression of PHD2 (P<0.05). Conclusions Ischemia preconditioning may exert protection against renal ischemia reperfusion injury by inhibiting PHD2.