RMT1-10体外诱导耐受性树突状细胞对小鼠高危角膜移植排斥反应的抑制作用及其机制  

Inhibitory effect of RMT1-10-induced tolerogenic dendritic cells in vitro on high-risk corneal allograft rejection in mice and its mechanism

作　　者：赵敏 杨柳清 王梦宇 陶钰 郭永越 苏锐锋[1] 石晶[1] 谭小波[1] Zhao Min;Yang Liuqing;Wang Mengyu;Tao Yu;Guo Yongyue;Su Ruifeng;Shi Jing;Tan Xiaobo(Department of Ophthalmology,the Affiliated Hospital of Chengde Medical University,Chengde 067000,China;Department of Ophthalmology,Chengde Central Hospital,Chengde 067000,China)

机构地区：[1]承德医学院附属医院眼科,承德067000 [2]承德市中心医院眼科,承德067000

出　　处：《中华实验眼科杂志》2022年第8期725-733,共9页Chinese Journal Of Experimental Ophthalmology

基　　金：河北省自然科学基金面上项目(H2015406054);河北省自然科学基金精准医学联合基金培育项目(H2020406019);河北省科技厅技术创新引导专项-科技工作会商项目;河北省硕士研究生创新资助项目(CXZZSS2021141)。

摘　　要：目的探讨RMT1-10体外诱导耐受性树突状细胞(Tol-DCs)对小鼠高危角膜移植排斥反应的抑制作用及其机制。方法选取SPF级雄性BALB/c小鼠100只和C57BL/6小鼠50只,获取C57BL/6小鼠骨髓来源的未成熟树突状细胞(imDCs),按照诱导干预不同分为imDCs组(不干预)、成熟树突状细胞(mDCs)组(加入脂多糖)、RMT1-10组(加入RMT1-10和脂多糖)和IgG同型对照组(加入IgG同型抗体和脂多糖),培养7 d后采用流式细胞术检测各组DCs表型CD11c、CD80、CD86、主要组织相容性复合物(MHC)-Ⅱ、T细胞免疫球蛋白和黏蛋白结构域分子(Tim)-4和CD103表达水平;采用酶联免疫吸附法测定细胞培养液上清液中白细胞介素10(IL-10)和转化生长因子β(TGF-β)质量浓度。建立混合淋巴细胞培养体系,采用细胞计数试剂盒8法检测各组DCs刺激CD4^(+)T细胞增生的刺激指数(SI)。以角膜基质缝线法诱导BALB/c小鼠角膜新生血管,以4个象限新生血管均匀长入角膜中周区的小鼠作为受体。将80只受体小鼠采用随机数字表法随机分为imDCs组、mDCs组、RMT1-10组和IgG同型对照组,每组20只,各组分别于尾静脉注射相应DCs(1×10^(6)个/100μl)。注射后7 d,以C57BL/6小鼠为供体行穿透角膜移植术。术后1个月,每日裂隙灯显微镜下观察受体小鼠角膜植片排斥体征,包括角膜混浊、水肿及新生血管。术后21 d,每组抽取5只受体小鼠,耳廓皮下注射20μl(1×10^(6)个细胞)正常C57BL/6小鼠脾脏细胞,24 h后测量耳廓肿胀度评价迟发型超敏反应(DTH)。结果RMT1-10组DCs中CD80、CD86、MHC-Ⅱ和Tim-4阳性细胞占CD11c阳性细胞百分比均明显低于mDCs组,差异均有统计学意义(均P<0.001);RMT1-10组Tim-4阳性细胞百分比明显低于imDCs组,CD103阳性细胞百分比明显高于其他3个组,差异均有统计学意义(均P<0.001)。RMT1-10组细胞培养上清液中IL-10和TGF-β质量浓度明显高于其他组,差异均有统计学意义(均P<0.001)。各组DCs�Objective To investigate the inhibitory effect of RMT1-10-induced tolerogenic dendritic cells(Tol-DCs)in vitro on high-risk corneal allograft rejection in mice and its mechanism.Methods One hundred SPF male BALB/c mice and fifty SPF male C57BL/6 mice were selected.Bone marrow-derived immature dendritic cells(imDCs)obtained from C57BL/6 mice were divided into imDCs group,mature dentritic cells(mDCs)group,RMT1-10 group,and IgG isotype control group.The imDCs in the four groups were cultured with no intervention,lipopolysaccharide,RMT1-10 and lipopolysaccharide,or IgG isotype antibody and lipopolysaccharide for 7 days according to grouping.The expression levels of different phenotypes of DCs including CD11c,CD80,CD86,major histocompatibility complex(MHC)-Ⅱ,T cell immunoglobulin and mucin domain containing molecule(Tim)-4 and CD103 in the four groups were detected by flow cytometry.The concentrations of interleukin-10(IL-10)and transforming growth factor-β(TGF-β)in the DCs supernatants were determined by enzyme-linked immunosorbent assay.A mixed lymphocyte culture system was established,and the stimulation index(SI)of CD4^(+)T cell proliferation stimulated with DCs was detected by cell counting kit 8 method.Corneal neovascularization was induced by corneal stromal suture in BALB/c mice,and the 80 mice with neovascularization in 4 quadrants growing into the middle and peripheral cornea were used as recipients.The recipient mice were randomized into imDCs group,mDCs group,RMT1-10 group,and IgG isotype control group using the random number table method,with 20 mice in each group.An injection of corresponding DCs(1×10^(6)cells/100μl)was administered to the recipient mice via the tail vein according to grouping.At 7 days following the injection,C57BL/6 mice were used as donors and penetrating keratoplasty was performed.Within one month after the operation,signs of corneal grafts rejection,including opacity,edema and neovascularization,were observed by slit lamp biomicroscopy and scored every day.At 21 days after the

关 键 词：角膜移植 移植排斥 免疫耐受 树突状细胞 单克隆抗体 TIM-1 RMT1-10 

分 类 号：R779.65[医药卫生—眼科]