中华实用儿科临床杂志
中華實用兒科臨床雜誌
중화실용인과림상잡지
Journal of Applied Clinical Pediatrics
2015年
9期
711-714
,共4页
倪佳佳%郭燕%杨玲云%王荣%林加娟%丁桂霞%张爱华%黄松明
倪佳佳%郭燕%楊玲雲%王榮%林加娟%丁桂霞%張愛華%黃鬆明
예가가%곽연%양령운%왕영%림가연%정계하%장애화%황송명
顺铂%线粒体功能%microRNA-709%肾小管上皮细胞损伤
順鉑%線粒體功能%microRNA-709%腎小管上皮細胞損傷
순박%선립체공능%microRNA-709%신소관상피세포손상
Cisplatin%Mitochondrial function%MicroRNA-709%Renal tubular epithelial cell injury
目的 探讨microRNA-709(miR-709)在顺铂引起肾小管上皮细胞损伤模型中的作用及机制.方法 体外培养小鼠肾小管上皮细胞,予不同浓度(0、1、5、10、20 μmol/L)顺铂刺激24 h,以10 μmol/L顺铂刺激不同时间(0、2、6、12、24 h).采用实时荧光定量PCR(RT-PCR)技术检测细胞miR-709的表达变化.实验组分为上调对照组、miR-709过表达组、下调对照组、miR-709下调组,其中miR-709下调组和下调对照组加顺铂刺激.应用Annexin V-FITC/PI双染法检测细胞凋亡,分光光度法检测含半肮氨酸的天冬氨酸水解酶(Caspase-3)活性,Western blot法和RT-PCR检测上皮细胞标志蛋白E-cadherin及其mRNA的变化.JC-1(线粒体膜电位)荧光探针检测线粒体膜电位,流式细胞术检测线粒体超氧化物(mitoSOX)生成,PCR检测线粒体基因(mtDNA)拷贝数.结果 miR-709在5 μmol/L顺铂刺激小管上皮细胞后开始明显升高(F =22.17,P＜0.05),10μmol/L顺铂刺激12 h开始明显升高(F=33.462,P＜0.05);过表达miR-709后细胞凋亡数目、Caspase-3活性较上调对照组增加,E-cadherin表达较上调对照组下降,差异均有统计学意义(凋亡:1.54±0.20比1.00±0.23;Caspase-3活性:1.27±0.08比0.97 ±0.08;E-cadherin:0.47 ±0.15比1.00±0.10;t=-3.086、-5.882、5.671,P均＜0.05);线粒体功能指标线粒体膜电位、mtDNA拷贝数较对照组降低,mitoSOX生成较对照组增加,差异均有统计学意义(JC-1:0.80±0.04比1.05±0.08;mtDNA:0.58 ±0.15比1.00±0.75;mitoSOX:1.31±0.16比1.00±0.05;t =4.687、4.943、-3.694,P均＜0.05).而顺铂刺激后,miR-709下调组细胞凋亡数目、Caspase-3活性较下调对照组降低,E-cadherin表达较下调对照组增高,差异均有统计学意义(凋亡:1.35±0.10比1.86±0.44;Caspase-3活性:1.04±0.12比1.30±0.09;E-cadherin:0.86±0.08比0.54±0.05;F=18.489、20.932、33.323,P均＜0.05);线粒体膜电位、mtDNA拷贝数较对照组增加,mitoSOX生成较下调对照组降低,差异均有统计学意义(JC-1:0.94±0.06比0.75±0.05;mtDNA:0.68±0.09 比0.27±0.12;mitoSOX:0.91 ±0.09比1.22 ±0.08;F =21.726、59.330、23.813,P均＜0.05).结论 miR-709可能通过负性调控线粒体功能参与顺铂引起的肾小管上皮细胞损伤.
目的 探討microRNA-709(miR-709)在順鉑引起腎小管上皮細胞損傷模型中的作用及機製.方法 體外培養小鼠腎小管上皮細胞,予不同濃度(0、1、5、10、20 μmol/L)順鉑刺激24 h,以10 μmol/L順鉑刺激不同時間(0、2、6、12、24 h).採用實時熒光定量PCR(RT-PCR)技術檢測細胞miR-709的錶達變化.實驗組分為上調對照組、miR-709過錶達組、下調對照組、miR-709下調組,其中miR-709下調組和下調對照組加順鉑刺激.應用Annexin V-FITC/PI雙染法檢測細胞凋亡,分光光度法檢測含半骯氨痠的天鼕氨痠水解酶(Caspase-3)活性,Western blot法和RT-PCR檢測上皮細胞標誌蛋白E-cadherin及其mRNA的變化.JC-1(線粒體膜電位)熒光探針檢測線粒體膜電位,流式細胞術檢測線粒體超氧化物(mitoSOX)生成,PCR檢測線粒體基因(mtDNA)拷貝數.結果 miR-709在5 μmol/L順鉑刺激小管上皮細胞後開始明顯升高(F =22.17,P＜0.05),10μmol/L順鉑刺激12 h開始明顯升高(F=33.462,P＜0.05);過錶達miR-709後細胞凋亡數目、Caspase-3活性較上調對照組增加,E-cadherin錶達較上調對照組下降,差異均有統計學意義(凋亡:1.54±0.20比1.00±0.23;Caspase-3活性:1.27±0.08比0.97 ±0.08;E-cadherin:0.47 ±0.15比1.00±0.10;t=-3.086、-5.882、5.671,P均＜0.05);線粒體功能指標線粒體膜電位、mtDNA拷貝數較對照組降低,mitoSOX生成較對照組增加,差異均有統計學意義(JC-1:0.80±0.04比1.05±0.08;mtDNA:0.58 ±0.15比1.00±0.75;mitoSOX:1.31±0.16比1.00±0.05;t =4.687、4.943、-3.694,P均＜0.05).而順鉑刺激後,miR-709下調組細胞凋亡數目、Caspase-3活性較下調對照組降低,E-cadherin錶達較下調對照組增高,差異均有統計學意義(凋亡:1.35±0.10比1.86±0.44;Caspase-3活性:1.04±0.12比1.30±0.09;E-cadherin:0.86±0.08比0.54±0.05;F=18.489、20.932、33.323,P均＜0.05);線粒體膜電位、mtDNA拷貝數較對照組增加,mitoSOX生成較下調對照組降低,差異均有統計學意義(JC-1:0.94±0.06比0.75±0.05;mtDNA:0.68±0.09 比0.27±0.12;mitoSOX:0.91 ±0.09比1.22 ±0.08;F =21.726、59.330、23.813,P均＜0.05).結論 miR-709可能通過負性調控線粒體功能參與順鉑引起的腎小管上皮細胞損傷.
목적 탐토microRNA-709(miR-709)재순박인기신소관상피세포손상모형중적작용급궤제.방법 체외배양소서신소관상피세포,여불동농도(0、1、5、10、20 μmol/L)순박자격24 h,이10 μmol/L순박자격불동시간(0、2、6、12、24 h).채용실시형광정량PCR(RT-PCR)기술검측세포miR-709적표체변화.실험조분위상조대조조、miR-709과표체조、하조대조조、miR-709하조조,기중miR-709하조조화하조대조조가순박자격.응용Annexin V-FITC/PI쌍염법검측세포조망,분광광도법검측함반항안산적천동안산수해매(Caspase-3)활성,Western blot법화RT-PCR검측상피세포표지단백E-cadherin급기mRNA적변화.JC-1(선립체막전위)형광탐침검측선립체막전위,류식세포술검측선립체초양화물(mitoSOX)생성,PCR검측선립체기인(mtDNA)고패수.결과 miR-709재5 μmol/L순박자격소관상피세포후개시명현승고(F =22.17,P＜0.05),10μmol/L순박자격12 h개시명현승고(F=33.462,P＜0.05);과표체miR-709후세포조망수목、Caspase-3활성교상조대조조증가,E-cadherin표체교상조대조조하강,차이균유통계학의의(조망:1.54±0.20비1.00±0.23;Caspase-3활성:1.27±0.08비0.97 ±0.08;E-cadherin:0.47 ±0.15비1.00±0.10;t=-3.086、-5.882、5.671,P균＜0.05);선립체공능지표선립체막전위、mtDNA고패수교대조조강저,mitoSOX생성교대조조증가,차이균유통계학의의(JC-1:0.80±0.04비1.05±0.08;mtDNA:0.58 ±0.15비1.00±0.75;mitoSOX:1.31±0.16비1.00±0.05;t =4.687、4.943、-3.694,P균＜0.05).이순박자격후,miR-709하조조세포조망수목、Caspase-3활성교하조대조조강저,E-cadherin표체교하조대조조증고,차이균유통계학의의(조망:1.35±0.10비1.86±0.44;Caspase-3활성:1.04±0.12비1.30±0.09;E-cadherin:0.86±0.08비0.54±0.05;F=18.489、20.932、33.323,P균＜0.05);선립체막전위、mtDNA고패수교대조조증가,mitoSOX생성교하조대조조강저,차이균유통계학의의(JC-1:0.94±0.06비0.75±0.05;mtDNA:0.68±0.09 비0.27±0.12;mitoSOX:0.91 ±0.09비1.22 ±0.08;F =21.726、59.330、23.813,P균＜0.05).결론 miR-709가능통과부성조공선립체공능삼여순박인기적신소관상피세포손상.
Objective To investigate the role of microRNA-709 (miR-709) in murine renal tubular epithelial cells with Cisplatin treatment and its underlying mechanism.Methods The cells were cultured with Cisplatin in the concentrations (0,1,5,10,20 μmol/L) for 24 h in vitro,and underwent 10 μmol/L Cisplatin stimulation at different time points (0,2,6,12,24 h).Real-time fluorescence quantitative polymerase chain reaction (RT-PCR) was used to investigate the level of miR-709.mPTC were divided into a negative control group,miR-709 mimic group,inhibitor negative control (INNC) group,miR-709 inhibitor group,INNC group and miR-709 inhibitor group were treated with Cisplatin treatment.Annexin V-FITC/PI double staining was applied to detect apoptosis.Caspase-3 activity was detected by spectrophotometry.The mRNA and protein levels of E-cadherin were detected by RT-PCR and Western blot.Mitochondrial membrane potential and mitochondrial superoxide (mitoSOX) generation were detected by flow cytometry.Mitochondrial DNA(mtDNA) copy number was verified by PCR.Results The level of miR-709 increased in Cisplatin 5 μmol/L stimulation group (F =22.17,P ＜ 0.05) and increased further 12 h later after cultured in 10 μmol/L Cisplatin compared with that of the control group (F =33.462,P ＜ 0.05).After overexpression of miR-709,apoptotic rate and Caspase-3 activity of the mPTC increased,while the expression of E-cadherin declined when compared with that in the negative control group (apoptotic rate:1.54 ± 0.20 vs 1.00 ± 0.23;Caspase-3 activity:1.27 ± 0.08 vs 0.97 ± 0.08;E-cadherin:0.47 ± 0.15 vs 1.00 ± 0.10;t =-3.086,-5.882,5.671,all P ＜0.05).In addition,the levels of JC-1 and mtDNA decreased while mitoSOX increased compared with that of the control group (JC-1:0.80±0.04 vs 1.05 ±0.08;mtDNA:0.58 ±0.15 vs 1.00 ±0.75;mitoSOX:1.31 ±0.16 vs 1.00 ± 0.05;t =4.687,4.943,-3.694,all P ＜ 0.05).Downregulation of miR-709 attenuated the tubular cell injury and the mitochondrial dysfunction was induced by Cisplatin (apoptotic rate:1.35 ±0.10 vs 1.86 ±0.44;Caspase-3 activity:1.04 ±0.12 vs 1.30 ±0.09;E-cadherin:0.86 ±0.08 vs 0.54 ±0.05;JC-1:0.94 ±0.06 vs 0.75 ±0.05;mtDNA:0.68 ±0.09 vs 0.27 ±0.12;mitoSOX:0.91 ±0.09 vs 1.22 ±0.08;F =18.489,20.932,33.323,21.726,59.330,23.813,all P ＜ 0.05).Conclusion Mitochondrial function negatively regulated by miR-709 may be involved in the renal tubular epithelial cell injury induced by Cisplatin.
