浓缩生长因子对氧化应激状态下人牙髓干细胞生物学性能的影响  

Effect of concentrated growth factor on the biological performance of human dental pulp stem cells under oxidative stress status

作　　者：陈慧 张昊 赵雪纯 何俐 Chen Hui;Zhang Hao;Zhao Xuechun;He Li(Department of Stomatology,Renmin Hospital,Hubei University of Medicine,Shiyan 442000,China)

机构地区：[1]十堰市人民医院(湖北医药学院附属人民医院)口腔科,十堰442000

出　　处：《中华口腔医学杂志》2025年第2期151-159,共9页Chinese Journal of Stomatology

基　　金：湖北省卫生健康科研基金(WJ2021M054);湖北医药学院研究生科技创新项目(YC2023056)。

摘　　要：目的探讨浓缩生长因子(CGF)对过氧化氢(H_(2)O_(2))诱导的氧化应激状态下人牙髓干细胞(hDPSC)生物学性能的影响。方法采用组织块分离法从因正畸拔除的健康恒牙中提取hDPSC, 流式细胞术检测hDPSC的表面标志物CD34、CD45、CD90、CD105, 碱性磷酸酶(ALP)、茜素红S、油红O染色和集落形成实验对hDPSC进行鉴定。细胞计数试剂盒(CCK-8)检测后选用最适H_(2)O_(2)浓度构建hDPSC氧化应激模型。采用反复冻融法制备CGF条件培养基, CCK-8检测后选用最适CGF浓度用于后续实验。将hDPSC分为对照组、H_(2)O_(2)组(H_(2)O_(2)单独处理)、H_(2)O_(2)+CGF组(H_(2)O_(2)联合CGF处理)和CGF组(CGF单独处理), 细胞分组后进行后续所有实验。应用活性氧、β-半乳糖苷酶染色和蛋白质印迹法对氧化应激模型进行验证。CCK-8和细胞划痕实验分别检测CGF对氧化应激状态下hDPSC增殖和迁移能力的影响;ALP及茜素红S染色检测CGF对氧化应激状态下hDPSC成骨分化能力的影响;实时荧光定量PCR(RT-qPCR)检测成牙相关基因mRNA的表达, 蛋白质印迹法检测成牙、成骨相关蛋白的表达。结果分离培养的hDPSC阳性表达间充质干细胞表面标志物CD90、CD105, 阴性表达造血干细胞表面标志物CD34、CD45;hDPSC具有成骨、成脂向分化能力和克隆形成能力。200 μmol/L H_(2)O_(2)为构建氧化应激模型的最适浓度。20%CGF为用于后续实验的最适CGF浓度。与对照组相比, H_(2)O_(2)组衰老蛋白p53表达从(0.82±0.12)显著上调至(1.19±0.14)(P<0.05), β-半乳糖苷酶染色加深、活性氧荧光强度增加。H_(2)O_(2)+CGF组在第1、3、5、7天的增殖能力(0.23±0.01、0.50±0.02、1.60±0.07、1.80±0.21)均显著高于H_(2)O_(2)组(0.15±0.01、0.14±0.02、0.50±0.03、0.90±0.09)(均P<0.05);且H_(2)O_(2)+CGF组在12、24 h的细胞愈合能力[(47±7)%、(58±44)%]也均显著高于H_(2)O_(2)组[(36±2)%、(44±2)%](均P<0.05), ALP活性和矿化结节形成增加ObjectiveTo investigate the effect of concentrated growth factor(CGF)on the biological performance of human dental pulp stem cells(hDPSCs)under oxidative stress status induced by hydrogen peroxide(H_(2)O_(2)).MethodsThe hDPSCs were isolated by using tissue block separation method from healthy permanent teeth extracted for orthodontic reason.hDPSCs surface markers CD34,CD45,CD90 and CD105 were detected by flow cytometry.Alkaline phosphatase(ALP),alizarin red S(ARS),oil red O staining and colony formation assay were used to identify hDPSCs.After the cell counting kit-8(CCK-8)detection,the optimal H_(2)O_(2) concentration was used to construct the hDPSCs oxidative stress model.CGF conditioned medium was prepared by repeated freeze-thaw methods.After CCK-8 detection,the optimum CGF concentration was chosen for the subsequent experiments.The hDPSCs were divided into control group,H_(2)O_(2)(only H_(2)O_(2) processing),H_(2)O_(2)+CGF group(H_(2)O_(2) processing in combination with the CGF)and CGF group(only CGF processing).Subsequent experiments were performed according to these groups.The oxidative stress model was verified by reactive oxygen species,β-galactosidase staining and Western blotting.The effects of CGF on the proliferation and migration of hDPSCs under oxidative stress status were detected by CCK-8 and cell scratch assay,respectively.ALP activity and ARS staining were used to detect the effect of CGF on the osteogenic differentiation of hDPSCs under oxidative stress status.The mRNA expression levels of odontogenesis related genes were detected by real-time fluorescence quantitative PCR(RT-qPCR),and the expression levels of odontogenesis and osteogenesis related proteins were detected by Western blotting.ResultsIsolated hDPSCs showed positive expression of mesenchymal stem cells surface markers of CD90,CD105,and negative expression of hematopoietic stem cells surface markers CD34,CD45.The hDPSCs were proved to have the capacity of osteogenic,adipogenic differentiation and clone formation.The optimal concen

关 键 词：牙髓 牙髓干细胞 浓缩生长因子 过氧化氢 氧化应激 组织工程学 

分 类 号：R78[医药卫生—口腔医学]