富含半胱氨酸蛋白61对慢性肾脏病小鼠骨骼肌萎缩的影响及作用机制研究  

Role of cysteine-rich protein 61 in skeletal muscle atrophy of mice with chronic kidney disease

作　　者：林幼幼 左一丹 徐笛艳 古亮 胡菲菲 苏震[2] LIN Youyou;ZUO Yidan;XU Diyan;GU Liang;HU Feifei;SU Zhen(Department of Nephrology,Taizhou Hospital of Zhejiang Province(Taizhou Hospital Affiliated to Wenzhou Medical University),Taizhou 317000,China)

机构地区：[1]浙江省台州医院(温州医科大学附属台州医院)肾内科,317000 [2]温州医科大学附属第一医院肾内科

出　　处：《浙江医学》2025年第2期157-162,172,I0004,共8页Zhejiang Medical Journal

基　　金：国家自然科学基金项目(81671403、30871179);浙江省卫生健康委员会面上项目(2021KY203);温州市基础性科研项目(Y20220032)。

摘　　要：目的探讨富含半胱氨酸蛋白61(CCN1)对慢性肾脏病(CKD)小鼠骨骼肌萎缩的影响及作用机制,为CKD骨骼肌萎缩的治疗寻找新的靶点。方法将24只SPF级雄性小鼠按随机数字表法分为造模组、假手术组、过表达造模组、过表达假手术组,每组6只。造模组与过表达造模组经5/6肾切除术建立CKD小鼠模型,假手术组与过表达假手术组进行假手术。采用腺相关病毒(AAV9)载体传递系统过表达CCN1,采用自身对照,过表达造模组与过表达假手术组分别注射CCN1载体形成载体造模组与载体假手术组,分别注射空AAV9载体形成空载体造模组与空载体假手术组。过表达造模完成4周后测定各组小鼠体质量,采用小鼠代谢笼留取24 h尿量,采用颈椎脱臼法处死小鼠,采集小鼠腓肠肌、胫骨前肌、肾脏、血液标本。计算腓肠肌体质量比值与胫骨前肌体质量比值;马松染色观察肾脏病理改变;按试剂盒说明操作测定血肌酐、血尿素氮水平;层粘连蛋白免疫荧光染色观察胫骨前肌肌纤维横截面积及分布;反转录聚合酶链反应检测腓肠肌CCN1转录水平表达;采用蛋白质印迹法检测小鼠腓肠肌组织CCN1、肿瘤蛋白53(p53)、多肿瘤抑制因子1(p16)、细胞周期蛋白依赖性激酶抑制因子p21(p21)、视网膜母细胞瘤(RB)蛋白表达情况。结果与假手术组比较,造模组腓肠肌体质量比值下降,空载体造模组腓肠肌体质量比值、胫骨前肌体质量比值上升,载体造模组腓肠肌体质量比值上升、胫骨前肌体质量比值下降,差异均有统计学意义(均P<0.05);与空载体造模组比较,载体造模组腓肠肌体质量比值和胫骨前肌体质量比值均明显降低(均P<0.05);与空载体假手术组比较,载体假手术组胫骨前肌体质量比值明显降低(P<0.05)。与假手术组比较,造模组、空载体假手术组、空载体造模组、载体造模组胫骨前肌肌纤维平均横截面积均明显缩小(均P<0.0Objective To investigate the impact and mechanism of cysteine-rich protein 61(CCN1)on skeletal muscle atrophy in mice with chronic kidney disease(CKD)so as to explore new therapeutic targets for CKD-associated skeletal muscle atrophy.Methods Twenty-four SPF-grade male mice were randomly divided into a model group,a sham surgery group,an overexpression model group,and an overexpression sham group,with six mice in each group.The model and overexpression model groups underwent 5/6 nephrectomy to establish the CKD mouse model,while the sham and overexpression sham groups underwent sham surgery.An adeno-associated virus type 9(AAV9)vector delivery system was used to overexpress CCN1.By using self-controls,the overexpression model and overexpression sham surgery groups were injected with CCN1 vectors to form vector model and vector sham surgery groups,respectively,and they were injected with empty AAV9 vectors to form empty vector model and empty vector sham surgery groups,respectively.Four weeks after overexpression modeling,body weight of mice was measured in each group.Mice were housed in metabolic cages to collect 24-hour urine samples.Mice were sacrificed to collect gastrocnemius,tibialis anterior,kidney,and blood samples.The gastrocnemius-to-body weight ratio and tibialis anterior-to-body weight ratio were calculated.Renal pathological changes were observed via Masson's trichrome staining.Blood creatinine and blood urea nitrogen levels were determined according to kit instructions.Laminin immunofluorescence staining was used to observe the cross-sectional area and distribution of tibialis anterior muscle fibers.Reverse transcription pdymerase chain reaction was used to detect the transcriptional expression of CCN1 in the gastrocnemius.Western blotting was employed to detect the expression levels of proteins(CCN1,tumor protein 53,multiple tumor suppressor 1,cyclin-dependent kinase inhibitor 1A,retinoblastoma)in mouse gastrocnemius tissue.Results Compared with the sham surgery group,the gastrocnemius-to-body weight

关 键 词：慢性肾脏病 富含半胱氨酸蛋白61 骨骼肌萎缩 衰老 

分 类 号：R692[医药卫生—泌尿科学]