摘要
目的:探究过表达E26转化特异性变异体2(ETV2)在牙髓干细胞(DPSCs)成骨分化中的分子调控作用,阐明其对成骨细胞特异性转录因子Osterix(OSX)转录活性和巨噬细胞极化的影响机制。方法:构建ETV2过表达DPSCs稳定转染细胞系,通过转录组测序分析基因表达谱变化;采用双荧光素酶报告基因实验验证ETV2过表达对OSX启动子的直接调节作用,同时免疫荧光染色检测OSX蛋白表达。建立转染ETV2过表达DPSCs与巨噬细胞共培养体系,使用流式细胞术检测巨噬细胞CD86(M1型标志物)和CD206(M2型标志物)阳性率,实时荧光定量PCR检测炎症相关细胞因子白细胞介素(IL)-1β、IL-6、IL-1受体拮抗剂(IL-1ra)和转化生长因子-β(TGF-β)基因水平。将ETV2过表达DPSCs/支架复合物植入大鼠临界尺寸颅骨缺损区域,免疫荧光染色验证过表达ETV2对骨组织OSX和巨噬细胞标志物诱导型一氧化氮合酶(iNOS)和CD206表达的调节作用。结果:转录组分析显示,过表达ETV2显著激活了DPSCs成骨分化相关信号通路。双荧光素酶实验证实ETV2能够直接结合OSX启动子并激活其转录活性(P<0.001),免疫荧光染色发现ETV2过表达后DPSCs中OSX蛋白表达增加(P<0.01)。共培养实验发现,过表达ETV2显著调节巨噬细胞极化,CD86阳性细胞减少(P<0.01),CD206阳性细胞增加(P<0.05);同时,促炎因子IL-1β和IL-6基因表达下调(P<0.05),抗炎因子IL-1ra和TGF-β基因表达上调(P<0.01)。体内实验显示,过表达ETV2后大鼠术后3天缺损区骨组织OSX阳性细胞比例显著升高(P<0.05),CD206表达增加,iNOS表达下降。结论:ETV2在DPSCs中发挥双重分子调节作用:直接激活OSX基因转录和促进巨噬细胞向M2表型极化。
Objective:To investigate the molecular regulatory role of E26 transformation-specific variant 2(ETV2)in osteogenic differentiation of dental pulp stem cells(DPSCs)and elucidate its mechanisms of action on osteoblast-specific transcription factor Osterix(OSX)transcriptional activity and macrophage polarization.Methods:Stable ETV2-overexpressing DPSC cell lines were constructed,and gene expression profile changes were analyzed by transcriptome sequencing.Dual-luciferase reporter assays were employed to validate the direct regulatory effect of ETV2 overexpression on the OSX promoter,and immunofluorescence staining was performed to detect OSX protein expression.A co-culture system of ETV2-overexpressing DPSCs and macrophages was established.Flow cytometry was used to detect the positive rates of macrophage markers CD86(M1 marker)and CD206(M2 marker).Quantitative real-time PCR was performed to measure mRNA levels of inflammation-related cytokines including interleukin-1β(IL-1β),interleukin-6(IL-6),interleukin-1 receptor antagonist(IL-1ra),and transforming growth factor-β(TGF-β).ETV2-overexpressing DPSCs/scaffold composites were implanted into rat critical-size calvarial defect sites,and immunofluorescence staining was used to verify the regulatory effects of ETV2 overexpression on OSX and macrophage marker expression including inducible nitric oxide synthase(iNOS)and CD206 in bone tissue.Results:Transcriptomic analysis revealed that ETV2 overexpression significantly activated osteogenic differentiation-related signaling pathways in DPSCs.Dual-luciferase assays confirmed that ETV2 could directly bind to the OSX promoter and activate its transcriptional activity(P<0.001).Immunofluorescence staining demonstrated increased OSX protein expression in DPSCs after ETV2 overexpression(P<0.01).In the co-culture experiments,ETV2 overexpression significantly modulated macrophage polarization,with CD86-positive cells decreased(P<0.01)and CD206-positive cells increased(P<0.05).Meanwhile,mRNA levels of pro-inflammatory cytokines IL-1βand IL-6 were significantly downregulated(P<0.05),while mRNA levels of anti-inflammatory factors IL-1ra and TGF-βwere significantly upregulated(P<0.01).In vivo experiments showed that 3 days post-surgery,the proportion of OSX-positive cells in the bone tissue of the defect area was significantly higher in the ETV2 overexpression group(P<0.05),with increased CD206 expression and decreased iNOS expression.Conclusions:ETV2 exerts dual molecular regulatory functions in DPSCs:directly activating OSX gene transcription and promoting macrophage polarization toward the M2 phenotype.
作者
郜康
杜浩然
徐一帆
李子潇
周建
GAO Kang;DU Haoran;XU Yifan;LI Zixiao;ZHOU Jian(Department of International Medical Center,Beijing Stomatological Hospital,Capital Medical University,Beijing 100070,China;Beijing Laboratory of Oral Health,Capital Medical University,Beijing 100069,China;Laboratory for Clinical Medicine,Capital Medical University,Beijing 100069,China;Department of Dental Implant Center,Beijing Stomatological Hospital,Capital Medical University,Beijing 100070,China;Laboratory for Oral and General Health Integration and Translation,Beijing TianTan Hospital,Capital Medical University,Beijing 100070,China)
出处
《口腔生物医学》
2025年第4期197-205,共9页
Oral Biomedicine
基金
国家自然科学基金(82170951,82470961,81741106)
中国医学科学院中央级公益性科研院所基本科研业务费专项资金资助(2023-JKCS-19)。
