调控自噬流对巨噬细胞粘附与迁移功能的影响  

Effects of autophagic flux regulation on the adhesion and migration of macrophages

作　　者：姜彧滕 赵宇[1] 朱小东[1] 刘玉秋[1] 郭银凤[1] 吴贝贝 张晓良[1] Jiang Yuteng;Zhao Yu;Zhu Xiaodong;Liu Yuqiu;Guo Yinfeng;Wu Beibei;Zhang Xiaoliang(Department of Nephrology,Zhongda Hospital Affiliated to Southeast University School of Medicine,Nanjing 210009,Jiangsu Province,China)

机构地区：[1]东南大学医学院东南大学附属中大医院肾内科,南京210009

出　　处：《中华肾病研究电子杂志》2018年第4期172-178,共7页Chinese Journal of Kidney Disease Investigation（Electronic Edition）

基　　金：国家自然科学基金(81570612);江苏省临床医学研究中心项目(BL2014080);江苏省普通高校研究生科研创新计划资助项目(SJCX17_0056);中央高校基本科研业务费专项基金(SJCX17_0056)

摘　　要：目的巨噬细胞浸润是包括糖尿病肾病(diabetic nephropathy,DN)在内的多种慢性肾脏疾病的重要组织病理特征。本研究通过调控巨噬细胞(RAW264.7)自噬流各阶段,探究其对巨噬细胞黏附迁移功能的影响。方法体内实验,建立糖尿病肾病大鼠模型,于12周末分别处死正常大鼠、DN组大鼠,病理染色观察肾脏病理改变,检测肾组织巨噬细胞标志物及自噬相关标志物表达。体外实验,检测正常与高糖(30 m M)条件下,巨噬细胞自噬体数量变化,LC3、Beclin-1(自噬相关标志物)、P62(自噬体清除指标)的表达,以及巨噬细胞粘附迁移数量。分别加用自噬溶酶体降解抑制剂氯喹(CQ)、自噬体生成激活剂雷帕霉素(RAPA),观察其对巨噬细胞自噬标记物表达及其粘附迁移功能的影响,电镜观察巨噬细胞自噬体数量与自噬溶酶体形态的变化。结果体内实验,DN大鼠肾脏损伤明显,肾小球体积增大,基底膜增厚,系膜基质增多,肾组织CD68(巨噬细胞标志物)、P62表达增加(t=3.35、t=16.27,P<0.05),LC3表达减少(t=51.12,P<0.05);体外实验,在高糖组,电镜观察发现自噬体数量较正常组减少,Western印迹与免疫荧光显示自噬相关蛋白LC3、Beclin-1表达降低,P62表达升高(t=27.02,t=45.56、t=32.71,P<0.05),巨噬细胞粘附和迁移数量增多(t=6.87、t=8.76,P<0.05)。用CQ处理后,电镜观察发现巨噬细胞自噬体溶酶体降解受到抑制,Western印迹与免疫荧光显示自噬相关蛋白LC3、Beclin-1表达降低,P62表达升高(t=14.64、t=12.45、t=8.57,P<0.05);CQ进一步促进高糖诱导的巨噬细胞黏附迁移数量增多(t=4.37、t=7.27,P<0.05);RAPA增加巨噬细胞自噬体数量,Western与免疫荧光显示RAPA提高了被高糖抑制的巨噬细胞自噬水平,[LC3、Beclin-1表达升高,P62表达降低(t=9.37、t=11.53,t=8.73;P<0.05)],减少高糖诱导的巨噬细胞黏附、迁移数量增多(t=4.16、t=5.74,P<0.05)。结论高糖抑制自噬水平,促进巨噬细胞黏附Objective Macrophage infiltration is an important histopathological feature of many chronic renal diseases including diabetic nephropathy (DN).By modulating the autophagic flux stages of macrophages (RAW264.7),this study investigated their impacts on the adhesion and migration capacities of macrophages.Methods DN rat models were established with streptozotocin.The rats were sacrificed at the end of 12 weeks after treatment.Pathological staining was made to observe renal pathological changes,and renal macrophage markers and autophagy-related markers were detected.Under the normal and high glucose (30 mM) conditions,the number of autophagosomes in RAW264.7 macrophages,the expressions of LC3,Beclin-1 (autophagy-associated marker),and P62 (autophagosome clearance marker),and the number of macrophages with adhesion and migration were measured.The autophagy lysosomal degradation inhibitor chloroquine (CQ) and the autophagosome-production-activating agent rapamycin (RAPA) were added,respectively,to observe the effects on expressions of autophagy markers as well as adhesion and migration of macrophages.The electron microscopy method was used to observe the number of autophagosomes in macrophages and the changes of autophagy lysosomes.Results DN rats showed obvious renal damage:increased glomerular volume,thickened basement membrane,increased mesangial matrix,increased renal expressions of CD68 (macrophage marker),and P62 (t=3.35,t=16.27,P <0.05),and decreased expression of LC3 (t=51.12,P<0.05).In the high glucose group of in vitro experiments,the electron microscopy showed that the number of autophagosomes was lower than that of the normal group;Western blot and immunofluorescence showed that the expressions of autophagy-related proteins LC3 and Beclin-1 were both decreased,while the expression of P62 was increased (t=27.02,t=45.56,t= 32.71,P<0.05),and the number of macrophages with adhesion and migration was increased (t=6.87,t=8.76,P<0.05);However,after treatment with CQ,the electron microscopic observation showed that

关 键 词：巨噬细胞 高糖 自噬流 粘附 迁移 

分 类 号：R692[医药卫生—泌尿科学]