人巨噬细胞极化对小鼠周细胞-肌成纤维细胞转分化的体外作用研究  

The study of effect of Human macrophage polarization on Mouse Pericyte-to-Myofibroblast Transition in vitro

作　　者：王子杰[1] 桂泽平 郑明 杭周 韩志坚[1] 陶俊[1] 居小兵[1] 谭若芸 顾民[1] Wang Zijie;Gui Zeping;Zheng Ming;Hang Zhou;Han Zhijian;Tao Jun;Ju Xiaobin;Tan Ruoyun;Gu Min(Department of Urology,the First Affiliated Hospital of Nanjing Medical University,Jiangsu 210029,China;Department of Urology,the Second Affiliated Hospital of Nanjing Medical University,Nanjing,Jiangsu 210003,China)

机构地区：[1]南京医科大学第一附属医院泌尿外科,南京210029 [2]南京医科大学第二附属医院泌尿外科,南京210003

出　　处：《中华器官移植杂志》2022年第6期346-351,共6页Chinese Journal of Organ Transplantation

基　　金：国家自然科学基金项目(81900684);江苏省自然科学基金青年基金项目(BK20191063)。

摘　　要：目的探究巨噬细胞极化对周细胞-肌成纤维细胞转分化及移植肾间质纤维化形成中的作用。方法分别收集5例2010~2015年在南京医科大学第一附属医院确诊慢性移植物失功(CGD)受者的移植肾组织和正常肾组织(对照组), 采用常规染色和免疫荧光染色的方法检测M1型和M2型巨噬细胞在肾组织中的表达与分布, 并用聚合酶链式反应(PCR)法检测样本中CD68、CD206和iNOS mRNA;采用转化生长因子-β1(TGF-β1)体外刺激小鼠血管周细胞系, 采用免疫印迹、细胞荧光的方法检测周细胞中α-平滑肌肌动蛋白(α-SMA)和血小板衍生生长因子受体-β(PDGFR-β)的表达;分别构建血管周细胞与M1型或M2型巨噬细胞联合培养模型, 采用免疫印迹、细胞荧光、PCR等方法检测周细胞中α-SMA和PDGFR-β的表达。结果在CGD受者的移植肾组织中观察到显著的CD68^(+)iNOS^(+)的M1型巨噬细胞浸润, 而CD68^(+)CD206^(+)细胞未显著浸润, 且CD68、iNOS、CD206 mRNA在CGD组中的表达均高于对照组(P<0.05);在CGD移植肾组织中, α-SMA和PDGFR-β的蛋白表达升高(P<0.05), 且α-SMA和PDGFR-β双染的细胞浸润于CGD受者移植肾间质中。体外实验中, TGF-β1可刺激小鼠周细胞中α-SMA和PDGFR-β升高(P<0.05), 且呈现时间依赖性;免疫印迹和细胞荧光中均可见M1型巨噬细胞可在体外促进小鼠周细胞中周细胞-肌成纤维细胞转分化相关指标升高, 而M2型巨噬细胞无法促进周细胞发生周细胞-肌成纤维细胞转分化过程。结论 M1型巨噬细胞极化可能通过促进周细胞-肌成纤维细胞转分化过程, 促进移植肾间质纤维化的形成。Objective To explore the role of macrophage polarization on pericyte-to-myofibroblast transition and renal allograft fibrosis after kidney transplantation(KT).Methods Allograft tissues were harvestedfrom recipients with chronic allograft dysfunction(CGD)and normal kidney tissues.The expression and distribution of M1/M2 macrophages in kidney tissues were detected by routine and immunofluorescent staining;mRNA of CD68,CD206 and iNOS detected by polymerase chain reaction(PCR);Murine vascular pericytes subjected to TGF-β1 in vitro and the expressions ofα-SMA and PDGFR-βin perivascular cells detected by immunoblotting and cellular fluorescence;The co-culturing models of vascular pericytes and M1/M2 macrophages were constructed.The expressions ofα-SMA and PDGFR-βin pericytes were detected by immunoblotting,cellular fluorescence and PCR.Results A marked infiltration of CD68^(+)iNOS^(+)M1 macrophages was present in allograft tissues of recipients with CGD while no obvious infiltration of CD68^(+)CD206^(+)was observed.The mRNA levels of CD68,iNOS and CD206 were significantly higher in CGD group than those in control group(P<0.05);In CGD allograft tissues,protein expressions ofα-SMA and PDGFR-βspiked markedly(P<0.05)while cells with double staining ofα-SMA and PDGFR-βwere markedly infiltrated in interstitial area of CGD allograft.TGF-β1 could induce a marked elevation of PMT-related markers in a time-dependent manner(P<0.05);Immunoblotting and cellukar fluorescence indicated that M1 macrophages could promote the elevations ofα-SMA and PDGFR-βin pericytes in vitro while M2 macrophages showed no effect on pericyte-to-myofibroblast transition in pericytes.Conclusions M1 macrophage polarization may promote the formation of renal allograft interstitial fibrosis through promoting PMT.

关 键 词：肾移植 巨噬细胞 慢性移植物失功 

分 类 号：R699.2[医药卫生—泌尿科学]