中介素对缺氧/复氧大鼠肾小管上皮细胞增殖作用及细胞周期的影响及其机制  

Research of the effect in intermedin on cell proliferation repair of renal tubular cell hypoxia-reoxygenation injury

作　　者：王艳红[1] 田继华[1] 苏晓乐[2] 乔晞[2] 李荣山[3] 

机构地区：[1]山西医科大学微生物学与免疫学教研室 [2]山西医科大学第二医院肾内科 [3]山西省人民医院肾内科,山西省肾脏病研究所,山西太原030001

出　　处：《中国药理学通报》2015年第4期482-487,共6页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No 30971380);山西医科大学博士启动基金(No 03201302);山西医科大学基础医学院331基金(No 201406);山西医科大学科技创新基金(No 01201403)

摘　　要：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。Aim To evaluate the effect of intermedin（ IMD） on cell proliferation and regeneration in rat tubular epithelial cell line（ NRK-52E） that was subjected to hypoxia-reoxygenation（ H / R） injury. Methods The NRK-52 E cells were divided into control group and three model groups（ H / R,H / R ＋ primitive vector,H / R ＋ IMD vector）. The content of LDH was detected 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 andprotein levels. Results ① In comparison to the control,H / R treatment decreased the cell viability and increased LDH activity（ P 0. 01）; in contrast,compared to H / R,IMD treatment ameliorated cell viability（ 79. 15 ± 1. 421% vs 61. 22 ± 1. 63%,P 〈0. 05） and decreased LDH activities by 33. 85%（ P 〈0. 01）. ②The proliferation of NRK-52 E cells was significantly inhibited by H / R treatment. In comparison to the control,H / R treatment of NRK-52 E 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/ M phase 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-regulated expression of cyclin D1,CDK4 and decreased the expression of p57 in NRK-52 E cells. ④Cyclin D1 had a predominantly nuclear localization in NRK-52Ecells,although cytoplasmic localization was also observed. Conclusion The study shows that the overexpression 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-regulation of p57.

关 键 词：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 

分 类 号：R332[医药卫生—人体生理学] R322.61[医药卫生—基础医学]