生长相关蛋白43通过抑制T细胞核因子1入核保护足细胞的机制  

Growth associated protein-43 protects podocytes by decreasing nuclear factor of activated T-cells cytoplasmic 1 expression

作　　者：连智雯 张丽[2] 窦曹帅 张鸿[2] 柯贵宝 陈雪芹 李卓[2] 马建超[2] 廖如意[2] 何朝生[2] 梁馨苓[2] 刘双信 LIAN Zhiwen;LI Zhuo;ZHANG li;DOU Caoshuai;ZHANG Hong;KE Guibao;CHEN Xueqin;MA Jianchao;LIAO Yuyi;HE Chaoshen;LIANG Xinling;LIU Shuangxin(The Second School of Clinical Medicine,Southern Medical University,Guangzhou 510080,China;Department of Nephrology,Guangdong Provincial People's Hospital,Guangdong Academy of Medical Sciences,Guangzhou 510080,China;School of Medicine,South China University of Technology,Guangzhou 510006,China)

机构地区：[1]南方医科大学第二临床医学院,广州510080 [2]广东省人民医院肾内科广东省医学科学院,广州510080 [3]华南理工大学医学院,广州510080

出　　处：《肾脏病与透析肾移植杂志》2019年第6期526-531,共6页Chinese Journal of Nephrology,Dialysis & Transplantation

基　　金：广州市科技计划项目(201707010009);国家自然科学基金面上项目(81670656,81870508);广东省博士后基金((2017M612624);广东省医学科学基金(A2017564);广东省自然科学基金项目(2018A030313260);广东省科技计划项目(2014A020209001,2015A020210069)

摘　　要：目的:探讨生长相关蛋白43(growth associated protein-43,GAP-43)通过抑制T细胞核因子1(nuclear factor of activated T-cells cytoplasmic 1,NFATc1)入核,从而保护足细胞的机制。方法:(1)通过免疫荧光染色及激光共聚焦显微镜,观察GAP-43在不同肾小球疾病患者足细胞中的表达情况。(2)体外培养小鼠永生化足细胞,用脂多糖(LPS)100μg/ml分别刺激足细胞0h、24h、48h、72h后,采用免疫荧光染色、实时荧光定量PCR(RT-PCR)和Western印迹检测GAP-43 mRNA和蛋白的表达。(3)通过Western印迹检测足细胞过表达GAP-43后对足细胞标志蛋白nephrin、钙调磷酸酶(calcineurin,CaN)及核内NFATc1蛋白表达的影响。(4)通过划痕实验观察足细胞的活动性。结果:(1)与正常肾组织相比,肾小球疾病患者肾组织足细胞GAP-43表达降低;(2)用LPS刺激足细胞,72h后GAP-43的表达降低最明显;(3)足细胞过表达GAP-43蛋白并用LPS刺激后,升高的CaN表达下降,NFATc1入核降低(P<0.05),降低的足细胞标志蛋白nephrin表达显著恢复(P<0.05)。(4)过表达GAP-43蛋白并用LPS刺激后,足细胞的活动性降低。结论:GAP-43在足细胞中表达,是足细胞的一个保护因子,可能通过参与CaN-NFAT信号通路,抑制NFATc1的入核来保护足细胞损伤。Objective:To explore the possible molecular mechanisms involved in podocyte injury protection caused by growth associated protein-43(GAP-43). Methodology:(1)Expression of GAP-43 in podocytes of patients with different glomerular diseases were observed by immunofluorescence staining and laser confocal microscopy.(2)Immortalized mouse podocytes was stimulated with lipopolysaccharide(LPS),expression of GAP-43 mRNA and protein were detected by Western blot,quantitative RT-PCR and immunofluorescence staining.(3)Immortalized mouse podocytes were overexpressed GAP-43 and were treated with LPS for 72 h before harvest.Western blot,quantitative RT-PCR and immunofluorescence staining were used to evaluate the expression of GAP-43,nephrin,calcineurin and nuclear factor of activated T cells c1(NFATc1).Using wound-healing assay,the migration in podocyte with overexpression GAP-43 were analyzed. Results:GAP-43 was decreased in LPS-treated podocytes in vitro.Increased nephrin,decreased calcineurin and nuclear NFATc1 was observed in LPS treated podocyte with overexpression of GAP-43;the migration ability was reduced after GAP-43 overexpressing. Conclusion:Our findings demonstrated that GAP-43 ameliorates podocyte injury by suppressing calcineurin/NFATc1 signaling,which may present a promising target for therapeutic intervention.

关 键 词：生长相关蛋白43 足细胞 凋亡 T细胞核因子1 

分 类 号：R692[医药卫生—泌尿科学]