淫羊藿苷预处理增强人牙周膜干细胞对M1型巨噬细胞的影响  

Icariin pretreatment enhances effect of human periodontal stem cells on M1-type macrophages

作　　者：喻婷 吕冬梅 邓浩 孙涛 程钎 Yu Ting;Lyu Dongmei;Deng Hao;Sun Tao;Cheng Qian(Luzhou Key Laboratory of Oral&Maxillofacial Reconstruction and Regeneration,Southwest Medical University,Luzhou 646000,Sichuan Province,China;Institute of Stomatology,Southwest Medical University,Luzhou 646000,Sichuan Province,China;Department of Orthodontics,Affiliated Stomatological Hospital of Southwest Medical University,Luzhou 646000,Sichuan Province,China;School of Stomatology,Southwest Medical University,Luzhou 646000,Sichuan Province,China)

机构地区：[1]西南医科大学口颌面修复重建和再生泸州市重点实验室,四川省泸州市646000 [2]西南医科大学口腔医学研究所,四川省泸州市646000 [3]西南医科大学附属口腔医院正畸科,四川省泸州市646000 [4]西南医科大学口腔医学院,四川省泸州市646000

出　　处：《中国组织工程研究》2025年第7期1328-1335,共8页Chinese Journal of Tissue Engineering Research

基　　金：泸州市科技计划项目(2023JYJ055),项目负责人:程钎;四川省医学青年创新科研课题计划(Q21053),项目负责人:程钎;西南医科大学附属口腔医学院导师组能力提升资助项目(2022DS14),项目负责人:程钎;西南医科大学大学生创新创业训练项目(S202210632302),项目负责人:邓浩。

摘　　要：背景:人牙周膜干细胞对M1型巨噬细胞促炎功能有一定抑制作用,具有抗炎等药理活性的淫羊藿苷是否能增强人牙周膜干细胞对M1型巨噬细胞的抑制作用尚不明确。目的:探究淫羊藿苷预处理人牙周膜干细胞后对M1型巨噬细胞的影响。方法:分离培养原代人牙周膜干细胞,并进行鉴定。对THP-1细胞进行诱导,采用免疫荧光染色及PCR进行M1型巨噬细胞鉴定。用含浓度10^(-7),10^(-6),10^(-5),10^(-4)mol/L淫羊藿苷的α-MEM完全培养基培养人牙周膜干细胞1,3,5,7 d,采用CCK-8法筛选合适的淫羊藿苷浓度进行实验。将α-MEM完全培养基、未处理的人牙周膜干细胞α-MEM条件培养基及淫羊藿苷预处理24 h的人牙周膜干细胞α-MEM条件培养基与RPMI-1640完全培养基以1∶1的比例对M1型巨噬细胞进行条件培养,分别为对照组、未处理组及预处理组,24 h后RT-PCR法检测M1型巨噬细胞炎症因子的mRNA表达情况;ELISA法检测M1型巨噬细胞炎症因子的蛋白表达情况;Western blot检测M1/M2型巨噬细胞表面标记物及核转录因子κB通路相关蛋白的表达。结果与结论:(1)CCK-8检测结果显示,10^(-7),10^(-6),10^(-5),10^(-4)mol/L淫羊藿苷对人牙周膜干细胞均无细胞毒性,且从第5天起,各浓度都提高了细胞活力,促进细胞增殖,选择10^(-4)mol/L淫羊藿苷进行后续实验。(2)RT-PCR法及ELISA检测结果显示,与对照组相比,未处理组及预处理组均降低了M1型巨噬细胞白细胞介素1β、白细胞介素6及肿瘤坏死因子α的表达与分泌(P<0.05),且预处理组低于未处理组(P<0.05)。(3)Western blot检测结果显示,与未处理组相比,预处理组CD86的表达明显降低(P<0.05);与对照组相比,未处理组和预处理组M2型巨噬细胞表面标志物CD206的表达均升高(P<0.01),且预处理组明显高于未处理组(P<0.01);M1型巨噬细胞经24 h条件培养后,与对照组相比,核转录因子κB/P65的表达在未处理组和预处理组均�BACKGROUND:Human periodontal stem cells have a certain inhibitory effect on the pro-inflammatory function of M1-type macrophages,and it is not clear whether icariin,which has anti-inflammatory and other pharmacological activities,can enhance the inhibitory effect of human periodontal stem cells on M1-type macrophages.OBJECTIVE:To investigate the effect of icariin on M1 macrophages after pretreatment of human periodontal stem cells.METHODS:Primary human periodontal stem cells were isolated,cultured and characterized.THP-1 was induced and M1-type macrophages were identified by immunofluorescence staining and PCR.Human periodontal stem cells were cultured withα-MEM complete medium containing concentrations of 10~(-7),10~(-6),10~(-5),and 10~(-4)mol/L icariin,and the cytotoxicity of Icariin on human periodontal stem cells was detected by the CCK-8 assay at 1,3,5,and 7 days,respectively.α-MEM complete medium,untreatedα-MEM conditioned medium for human periodontal stem cells andα-MEM conditioned medium for human periodontal stemcells pretreated with icariin for 24 hours were conditioned with RPMI-1640 complete medium in a 1:1 ratio for M1-type macrophages in the control,untreated,and pretreated groups,and 24 hours later,the mRNA expression of inflammatory factors in M1 macrophages was detected by RT-PCR.The protein expression of inflammatory factors in M1 macrophages was detected by ELISA.The expression of surface markers and nuclear factor-κB pathway-related proteins in M1/M2 macrophages was detected by western blot assay.RESULTS AND CONCLUSION:(1)CCK-8 assay results showed that 10~(-7),10~(-6),10~(-5),10~(-4)mol/L icariin was not cytotoxic to the human periodontal stem cells,and from day 5 onwards,all the concentrations increased the cell viability,and promoted the cell proliferation.10~(-4)mol/L icariin was selected for follow-up experiment.(2)RT-PCR and ELISA results showed that compared with the control group,the untreated group and the pretreated group both decreased the expression and secretion of interleuki

关 键 词：人牙周膜干细胞 淫羊藿苷 巨噬细胞 条件培养基共培养 表面标志物 炎症因子 抗炎 核转录因子κB信号通路 

分 类 号：R459.9[医药卫生—治疗学] R363[医药卫生—临床医学] R364