信号素4D在大鼠双膦酸盐相关颌骨坏死发生中的作用探讨  

The role of semaphorin 4D in the mechanism of bisphosphonate-related osteonecrosis of the jaw in rats

作　　者：潘巧婷 臧晓龙 孙照薇 潘梦琪 朱鑫美 李志勇 PAN Qiao-ting;ZANG Xiao-long;SUN Zhao-wei;PAN Meng-qi;ZHU Xin-mei;LI Zhi-yong(The Affiliated Hospital of Stomatology,School of Stomatology,Zhejiang University School of Medicine&Clinical Research Center for Oral Diseases of Zhejiang Province&Key Laboratory of Oral Biomedical Research of Zhejiang Province.Hangzhou 310006;Cancer Center of Zhejiang University,Hangzhou 310058;Department of Oral and Maxillofacial Surgery,Ningbo Stomatological College.Ningbo 315000,Zhejiang Province,China)

机构地区：[1]浙江大学医学院附属口腔医院·浙江大学口腔医学院,浙江省口腔疾病临床医学研究中心,浙江省口腔生物医学研究重点实验室,浙江杭州310006 [2]浙江大学癌症研究院,浙江杭州310058 [3]宁波口腔医院口腔颌面外科,浙江宁波315000

出　　处：《上海口腔医学》2022年第6期625-631,共7页Shanghai Journal of Stomatology

摘　　要：目的:研究双膦酸盐相关颌骨坏死(bisphosphonate-related osteonecrosis of the jaw,BRONJ)病损中信号素4D(semaphorin 4D,Sema4D)的表达变化,探索其在BRONJ发生过程中可能的作用。方法:采用唑来膦酸腹腔注射辅助拔牙构建BRONJ样大鼠模型,提取上颌骨标本行影像学及组织学检查,体外获取大鼠骨髓单核细胞(bone marrow mononuclear cells,BMMs)及骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)进行共培养,BMMs破骨向诱导后行细胞Trap染色及计数。在双膦酸盐(bisphosphonates,BPs)环境下,通过破骨定向诱导RAW264.7细胞,检测Sema4D表达差异。体外成骨向定向诱导MC3T3-E1细胞及BMSCs,BPs、Sema4D及Sema4D一抗干预后,检测细胞成骨、破骨相关基因ALP、Runx2和RANKL等的表达差异。采用GraphPad prism 8.0软件对数据进行统计学分析。结果:成功构建BRONJ样大鼠模型。拔牙后2周,实验组拔牙创愈合显著受限,颌骨暴露。H-E染色显示,实验组拔牙窝内新骨再生显著受限,可见死骨形成,拔牙窝软组织愈合受限。Trap染色显示,实验组拔牙窝附近破骨细胞数量显著少于对照组。显微CT扫描显示,实验组拔牙窝内骨密度及骨体积分数显著低于对照组。免疫组织化学染色结果显示,相比于对照组,实验组颌骨内Sema4D表达显著上升。体外研究显示,实验组BMMs破骨细胞向诱导能力显著低于对照组,BMSCs对破骨细胞诱导能力显著降低。破骨向诱导实验发现,BPs有效抑制破骨细胞形成,显著降低Sema4D表达。成骨向诱导实验发现,Sema4D显著降低成骨细胞Runx2、RANKL基因表达;加入Sema4D一抗后,ALP表达显著降低、RANKL表达显著上调。结论:BPs上调组织内Sema4D表达,干扰正常骨愈合时序,导致破骨-成骨偶联紊乱,抑制破骨细胞成熟,进而抑制成骨细胞分化和相关成骨因子表达,介导BRONJ发生。PURPOSE:To study the expression level of semaphorin 4D(Sema4D) in bisphosphonate-related osteonecrosis of the jaw(BRONJ) and to explore its possible role in the occurrence of BRONJ.METHODS:BRONJ-like rat model was established by intraperitoneal injection of zoledronic acid assisted with tooth extraction.The maxillary specimens were ex-tracted for imaging and histological examination,and bone marrow mononuclear cells(BMMs) and bone marrow mesenchy-mal stem cells(BMSCs) of each group were obtained in vitro for co-culture.Trap staining and counting were performed on monocytes after osteoclast induction.RAW264.7 cells were induced by osteoclast orientation under bisphosphonates(BPs)environment,and Sema4D expression was detected.Similarly,MC3T3-E1 cells and BMSCs were induced to osteogenic orientation in vitro,and the expression level of osteogenic and osteoclastic related genes ALP,Runx2,and RANKL was detected under the intervention of BPs,Sema4D and Sema4D antibody.Statistical analysis of the data was performed using GraphPad Prism 8.0 software.RESULTS:BRONJ-like rat model was successfully constructed.Two weeks after tooth extraction,the healing of the tooth extraction wound in the experimental group was significantly limited,and the tooth extraction wound was exposed.H-E staining results showed that regeneration of new bone in the extraction socket of the experimental group was significantly restricted,dead bone was formed,and the healing of the soft tissue was limited.The results of trap staining showed that the number of osteoclasts in the experimental group was significantly less than that in the control group.Micro-CT results showed that bone mineral density and bone volume fraction in the extraction socket of the experimental group were significantly lower than those of the control group.Immunohistochemical results showed that compared with the control group,the expression level of Sema4D in the experimental group was significantly increased.In vitro studies showed that compared with the control group,the oste

关 键 词：双膦酸盐相关颌骨坏死 双膦酸盐 信号素4D 骨髓单核细胞 破骨细胞 成骨细胞 

分 类 号：R782.3[医药卫生—口腔医学]