细胞衰老在老年小鼠急性肾损伤慢性化中的作用与机制研究  

Mechanism of cellular senescence in acute kidney injury that leads to chronic kidney disease in elderly mice

作　　者：王倩[1] 梁艳[1] 杨胜楠[1] 赵倩茹[1] 李冰[1] 李馨 田蕊[1] 刘章锁[2] 

机构地区：[1]郑州大学第一附属医院老年综合二科,450052 [2]郑州大学第一附属医院肾内科肾脏病研究所,450052

出　　处：《中华肾脏病杂志》2018年第1期44-51,共8页Chinese Journal of Nephrology

基　　金：国家自然科学基金（81300614）;河南省基础与前沿技术研究计划项目（142300410376）

摘　　要：目的探讨细胞衰老在老年小鼠缺血再灌注所致急性肾损伤慢性化中的作用及其机制。方法青年（8-10周龄）和老年（20～24月龄）C57BL／6雄性小鼠各16只，用小鼠缺血再灌注损伤模型构建急性肾损伤模型。随机数字表法分为4组：青年假手术（Young—Sham）组、老年假手术（Old—Sham）组、青年缺血再灌注损伤（Young—IRI）组、老年缺血再灌注损伤（Old．IRI）组。术后第1、3、7天称重并尾静脉取血，术后第14天处死小鼠，留取全血及双侧肾脏备检。检测血肌酐水平；肾组织标本用苏木精一伊红（HE）染色和天狼星红染色观察肾脏组织形态学改变；免疫荧光法检测I型胶原蛋白（Collagen—I）、巨噬细胞标志物F4／80及细胞周期阻滞标志物磷酸化组蛋白H3（p-HH3）和细胞核增殖抗原Ki67的表达；Western印迹法检测Collagen．I表达；实时荧光定量PCR法检测肿瘤坏死因子o【（TNF—O／_）、白细胞介素（IL）6、转化生长因子p（TGF—p）及Collagen．ImRNA的表达。结果与Sham组相比，Young—IRI组及Old．IRI组小鼠术后血肌酐均明显升高（P〈0．05）；与Young—IRI组相比，Old，IRI组术后各时间点血肌酐水平均显著增高（均Pc0．05）。肾组织病理结果提示，Young—IRI组小鼠术后第14天肾组织大部分损伤已修复；Old—Sham组肾组织有少量炎性细胞浸润及胶原沉积；Old。IRI组肾组织可见大量炎性细胞浸润及胶原沉积。免疫荧光结果显示，与Young—Sham组相比，Old—Sham组肾组织有散在F4／80阳性巨噬细胞浸润、Collagen—I沉积及部分p-HH3及Ki67双阳性的G2／M周期阻滞细胞，差异有统计学意义（均P〈0．05）；与Old—Sham组相比，Old—IRI组。肾组织上述各项指标相对表达量均显著增加（均P〈0．叭）。Western印迹结果显示，Old—Sham组Collagen—I表达量明显高于Young—Sham组（P〈0．05），Old—IRI组CollagenObjective To investigate the mechanism of cellular senescence in ischemia repeffusion injury （IRI）-induced acute kidney injury （AKI） that leads to chronic kidney disease （CKD） in elderly mice. Methods An acute kidney injury model was established in C57B1/6 male mice at ages 8- 10 weeks （young group） or 20- 24 months （old group） by bilateral IRI. The animals were randomly divided into 4 groups as follows： Young-Sham （n=8）, Old-Sham （n=8）, Young-IRI （n=8）, and Old-IRI groups （n=8）. All mice were weighted, and their blood was collected from the tail vein at days 1, 3, and 7 after surgery. The mice were killed on day 14 after surgery, and their kidneys were harvested for further analysis. Serum was used for the creatinine test. The changes of the renal tissue morphology and pathology were assessed using hematoxylin- eosin staining and sirius red staining. Immunofluoreseence staining of collagen ], F4/80, phosphor-histone H3 （p- HH3）, and Ki67 were performed to determine the stage of the collagen deposit, macrophage filtration, and cell cycle G2/M arrest. The collagen I expression was analyzed using western blot. The expression levels of TNF-ct, IL-6, TGF-I3, and collagen I were determined using real-time PCR. Results Compared with that in the sham group, the serum ereatinine levels in both Young- IRI and Old- IRI groups were obviously increased. The Young- IRI group recovered completely on day 7. The Old- IRI group had higher creatinine levels than the Young- IR] group at each time point. Morphology and pathology analyses revealed that acute injury was repaired in the Young-IR[ group, but slight inflammatory cell filtration and collagen deposition were observed in the Old- Sham and Old- IRI groups, respectively. Immunofluorescence staining revealed some F4/80-positive macrophage filtration, collagen I deposition, and p- HH3 and Ki67 double- positive nuclear tubular epithelial cells in the Old- Sham group, but considerably more positive results were found in the Old-IRI group.

关 键 词：再灌注损伤 细胞衰老 炎症 细胞周期阻滞 慢性肾脏病 

分 类 号：R692[医药卫生—泌尿科学]