CKLF1-C19多肽影响口腔黏膜下纤维性变中MFB活化及对SFRP表达的影响  

CKLF1-C19 polypeptide affects the activation of oral submucosal fibrotic myofibroblasts and the expression of secreted frizzled-related protein

作　　者：李艳莉 徐仰龙 邓金勇 何升腾 董方 LI Yanli;XU Yanglong;DENG Jinyong;HE Shengteng;DONG Fang(Department of Stomatology,Sanya Central Hospital,Sanya 572000,Hainan,China;Department of Endodontics,The Affiliated Stomatological Hospital of Zunyi Medical University,Zunyi 563003,Guizhou,China;Department of Stomatology,Sanya People's Hospital,Sanya 572000,Hainan,China)

机构地区：[1]三亚中心医院口腔科,海南三亚572000 [2]遵义医科大学附属口腔医院牙体牙髓科,贵州遵义563003 [3]三亚市人民医院口腔科,海南三亚572000

出　　处：《西部医学》2023年第2期195-202,共8页Medical Journal of West China

基　　金：海南省卫生健康行业科研项目(20A200142)。

摘　　要：目的探讨人趋化素样因子(CKLF1)C19多肽对口腔黏膜下纤维性变(OSF)中肌成纤维细胞(MFB)活化的作用以及对分泌型卷曲相关蛋白(sFRP)表达的影响。方法选取2020年1月~2021年1月在三亚中心医院口腔科门诊就诊并确诊为OSF的患者8例,并选择同期体检的健康志愿者8例,通过组织块培养法分别从健康志愿者和OSF患者颊粘膜组织分离成纤维细胞(FB)和MFB,免疫荧光染色检测平滑肌肌动蛋白(α-SMA)和纤维状肌动蛋白(F-actin)表达,以鉴定MFB;将MFB随机分为对照组、0.001 mg/L C19组、0.01 mg/L C19组、0.1 mg/L C19组,各浓度C19处理组分别使用含0.001、0.01、0.1 mg/LCKLF1-C19多肽培养液培养细胞48 h,对照组细胞正常培养,CCK-8法测定各时间点细胞增殖活性,收集上清后测定羟脯氨酸的含量,胶原凝胶收缩实验检测细胞收缩活性,Transwell小室实验检测细胞迁移与侵袭能力,实时荧光定量聚合酶链反应(qRT-PCR)和蛋白质免疫印迹(Western blot)实验测定细胞中sFRP1、sFRP2、sFRP5的mRNA与蛋白表达水平。结果分离的FB与MFB大多呈长梭形,MFB胞体较大、数目较多,且α-SMA呈阳性表达。与对照组比较,经过0.001、0.01、0.1 mg/L的CKLF1-C19多肽处理的MFB,在培养48 h和72 h后细胞增殖活性显著降低(P<0.05),上清中羟脯氨酸的含量减少(P<0.05),胶原凝胶收缩面积比率显著增加(P<0.05),迁移细胞数目与侵袭细胞数目均显著减少(P<0.05),细胞内sFRP1和sFRP2的mRNA和蛋白表达水平均显著上调(P<0.05),而sFRP5 mRNA和蛋白表达水平变化均无统计学意义(P>0.05)。结论CKLF1-C19多肽能够抑制口腔黏膜下纤维性变组织中肌成纤维细胞的增殖、迁移与侵袭,降低细胞的活化水平,并调控sFRP1和sFRP2的表达。Objective To explore the effect of human chemokine-like factor(CKLF1)C19 polypeptide on the activation of myofibroblasts in oral submucosal fibrosis(OSF)and the effect on the expression of secreted frizzled-related protein(sFRP).Methods Separate fibroblasts(FB)and myofibroblasts(MFB)from buccal mucosal tissues of healthy volunteers and OSF patients by tissue block culture method.Immunofluorescence staining to detect the expression ofα-SMA and F-actin to identify MFB.MFB were randomly divided into control group,0.001 mg/L C19 group,0.01 mg/L C19 group,0.1 mg/L C19 group,the C19 treatment group at each concentration was treated with a culture medium containing 0.001,0.01,0.1 mg/L of CKLF1-C19 polypeptide,and the cells were cultured for 48 h,control cells were cultured normally.The CCK-8 method measures the cell proliferation activity at each time point,after collecting the supernatant,determine the content of hydroxyproline,collagen gel contraction test detects cell contraction activity,Transwell chamber experiment to detect cell migration and invasion ability,real-time fluorescent quantitative polymerase chain reaction(qRT-PCR)and Western blot(Western blot)experiments were performed to determine the mRNA and protein expression levels of sFRP1,sFRP2 and sFRP5 in cells.Results The separated FBand MFB were mostly long fusiform.The MFB cell body was larger and more numerous,andα-SMA was positively expressed.Compared with the control group,MFB treated with CKLF1-C19 polypeptide at 0.001,0.01,and 0.1 mg/L showed a significant decrease in cell proliferation activity after 48 h and 72 h culture(P<0.05),the content of hydroxyproline in the supernatant decreased(P<0.05),the shrinkage area ratio of collagen gel increased significantly(P<0.05),the number of migrating cells and the number of invading cells were significantly reduced(P<0.05),the mRNA and protein expression levels of sFRP1 and sFRP2 in cells were significantly up-regulated(P<0.05),while the changes in sFRP5 mRNA and protein expression levels were not statistic

关 键 词：口腔黏膜下纤维性变 肌成纤维细胞 人趋化素样因子C19多肽 分泌型卷曲相关蛋白 

分 类 号：R781.5[医药卫生—口腔医学]