中国药理学通报
中國藥理學通報
중국약이학통보
CHINESE PHARMACOLOGICAL BULLETIN
2015年
4期
482-486,487
,共6页
王艳红%田继华%苏晓乐%乔晞%李荣山
王豔紅%田繼華%囌曉樂%喬晞%李榮山
왕염홍%전계화%소효악%교희%리영산
中介素%缺氧/复氧损伤%肾小管上皮细胞%细胞增殖%细胞周期%细胞周期蛋白
中介素%缺氧/複氧損傷%腎小管上皮細胞%細胞增殖%細胞週期%細胞週期蛋白
중개소%결양/복양손상%신소관상피세포%세포증식%세포주기%세포주기단백
intermedin%hypoxia-reoxygenation inju-ry%renal tubular epithelial cells%cell proliferation%cell cycle%cyclin
目的：探讨中介素（intermedin，IMD）对大鼠近端肾小管上皮细胞 NRK-52E 缺氧／复氧（H／R）后细胞增殖、细胞周期的影响。方法 NRK-52E 细胞随机分为对照组，模型组：缺氧／复氧组（H／R）、H／R ＋空质粒组、H／R ＋IMD 质粒组。MTT 法检测细胞增殖，比色法检测培养基上清 LDH 含量，流式细胞术检测细胞周期，Real time-PCR 法和 Western blot 法检测 cyclin D1、CDK、p57 mRNA 及蛋白表达，间接免疫荧光染色检测 cyclin D1亚细胞定位。结果①与对照组相比， H／R 组培养基中 LDH 含量升高了106％，同时细胞存活率明显下降，与 H／R 组比较，H／R ＋IMD 组培养基中 LDH 含量下降了33.85％（P ＜0.01），而细胞存活率增高（79.15％±1.42％ vs 61.22％±1.63％，P ＜0.05），②细胞周期结果显示，与对照组相比，H／R 组细胞 G0／G1期比例增加，S 期细胞比例降低（P ＜0.05）；与 H／R 组比较，H／R ＋IMD 组 G0／G1期细胞比例明显降低，而 S 及 G2期细胞比例增加（P ＜0.05）。③H／R 可增加 cyclin D1、CDK4及 p57的表达也增加（与对照组比较，P ＜0.05）；而 IMD 可进一步上调 cyclin D1、CDK4的表达，同时下调 p57的表达，与对照组及 H／R 组相比差异具有显著性（P ＜0.05）。④免疫荧光检测结果可见，cyclin D1呈红色荧光，在 NRK-52E 细胞内主要表达在细胞核中。结论IMD 可以上调 cyclin D1、CDK4蛋白表达，下调 p57的表达，促进细胞周期进展，从而加速肾组织 IRI后细胞增殖和修复。
目的：探討中介素（intermedin，IMD）對大鼠近耑腎小管上皮細胞 NRK-52E 缺氧／複氧（H／R）後細胞增殖、細胞週期的影響。方法 NRK-52E 細胞隨機分為對照組，模型組：缺氧／複氧組（H／R）、H／R ＋空質粒組、H／R ＋IMD 質粒組。MTT 法檢測細胞增殖，比色法檢測培養基上清 LDH 含量，流式細胞術檢測細胞週期，Real time-PCR 法和 Western blot 法檢測 cyclin D1、CDK、p57 mRNA 及蛋白錶達，間接免疫熒光染色檢測 cyclin D1亞細胞定位。結果①與對照組相比， H／R 組培養基中 LDH 含量升高瞭106％，同時細胞存活率明顯下降，與 H／R 組比較，H／R ＋IMD 組培養基中 LDH 含量下降瞭33.85％（P ＜0.01），而細胞存活率增高（79.15％±1.42％ vs 61.22％±1.63％，P ＜0.05），②細胞週期結果顯示，與對照組相比，H／R 組細胞 G0／G1期比例增加，S 期細胞比例降低（P ＜0.05）；與 H／R 組比較，H／R ＋IMD 組 G0／G1期細胞比例明顯降低，而 S 及 G2期細胞比例增加（P ＜0.05）。③H／R 可增加 cyclin D1、CDK4及 p57的錶達也增加（與對照組比較，P ＜0.05）；而 IMD 可進一步上調 cyclin D1、CDK4的錶達，同時下調 p57的錶達，與對照組及 H／R 組相比差異具有顯著性（P ＜0.05）。④免疫熒光檢測結果可見，cyclin D1呈紅色熒光，在 NRK-52E 細胞內主要錶達在細胞覈中。結論IMD 可以上調 cyclin D1、CDK4蛋白錶達，下調 p57的錶達，促進細胞週期進展，從而加速腎組織 IRI後細胞增殖和脩複。
목적：탐토중개소（intermedin，IMD）대대서근단신소관상피세포 NRK-52E 결양／복양（H／R）후세포증식、세포주기적영향。방법 NRK-52E 세포수궤분위대조조，모형조：결양／복양조（H／R）、H／R ＋공질립조、H／R ＋IMD 질립조。MTT 법검측세포증식，비색법검측배양기상청 LDH 함량，류식세포술검측세포주기，Real time-PCR 법화 Western blot 법검측 cyclin D1、CDK、p57 mRNA 급단백표체，간접면역형광염색검측 cyclin D1아세포정위。결과①여대조조상비， H／R 조배양기중 LDH 함량승고료106％，동시세포존활솔명현하강，여 H／R 조비교，H／R ＋IMD 조배양기중 LDH 함량하강료33.85％（P ＜0.01），이세포존활솔증고（79.15％±1.42％ vs 61.22％±1.63％，P ＜0.05），②세포주기결과현시，여대조조상비，H／R 조세포 G0／G1기비례증가，S 기세포비례강저（P ＜0.05）；여 H／R 조비교，H／R ＋IMD 조 G0／G1기세포비례명현강저，이 S 급 G2기세포비례증가（P ＜0.05）。③H／R 가증가 cyclin D1、CDK4급 p57적표체야증가（여대조조비교，P ＜0.05）；이 IMD 가진일보상조 cyclin D1、CDK4적표체，동시하조 p57적표체，여대조조급 H／R 조상비차이구유현저성（P ＜0.05）。④면역형광검측결과가견，cyclin D1정홍색형광，재 NRK-52E 세포내주요표체재세포핵중。결론IMD 가이상조 cyclin D1、CDK4단백표체，하조 p57적표체，촉진세포주기진전，종이가속신조직 IRI후세포증식화수복。
Aim To evaluate the effect of intermedin (IMD)on cell proliferation and regeneration in rat tu-bular epithelial cell line (NRK-52E)that was subjec-ted to hypoxia-reoxygenation (H/R)injury.Methods The NRK-52E cells were divided into control group and three model groups (H/R,H/R +primitive vec-tor,H/R +IMD vector).The content of LDH was de-tected to observe the influence of IMD on H/R injury. The cell proliferation was detected by MTT.The cell cycle was detected by flow cytometry.Real-time PCR and western blotting were used to determine mRNA and protein levels.Results ① In comparison to the con-trol,H/R treatment decreased the cell viability and in-creased LDH activity (P <0.01 );in contrast,com-pared to H/R,IMD treatment ameliorated cell viability (79.1 5 ±1 .421 % vs 61 .22 ±1 .63%,P <0.05)and decreased LDH activities by 33.85% (P <0.01 ).②The proliferation of NRK-52E cells was significantly in-hibited by H/R treatment.In comparison to the con-trol,H/R treatment of NRK-52E cells increased the proportion of cells in the G0 /G1 phase but decreased the proportion of cells in the S and G2 /M phases. Moreover,the over-expression of IMD resulted in S and G2 /Mphase redistribution and the accumulation of G2 /M-phase cells.The real-time PCR and western blotting results indicated that the mRNA and protein expression levels of cyclin D1 ,CDK4 and p57 were increased in H/R-treated cells.IMD further stimulated this up-reg-ulated expression of cyclin D1 ,CDK4 and decreased the expression of p57 in NRK-52E cells.④Cyclin D1 had a predominantly nuclear localization in NRK-52Ecells,although cytoplasmic localization was also ob-served.Conclusion The study shows that the over-expression of IMD may promote renal cell proliferation and regeneration after renal tubular cell H/R injury via the up-regulation of cyclin D1 ,CDK and the down-reg-ulation of p57.
