甲氨蝶呤对小鼠磨牙牙胚发育的影响  

Effect of methotrexate on the development in mouse molar tooth germ

作　　者：罗容春 韩雪 王海丞[1] 朱辙文 刘力维 王佐林[1] LUO Rongchun;HAN Xue;WANG Haicheng;ZHU Zhewen;LIU Liwei;WANG Zuolin(Shanghai Engineering Research Center of Tooth Restoration and Regeneration,Department of Oral Implantology,School and Hospital of Stomatology,Tongji University,Shanghai 200072,China)

机构地区：[1]上海牙组织修复与再生工程技术研究中心,同济大学口腔医学院,同济大学附属口腔医院口腔种植科,上海200072

出　　处：《口腔颌面外科杂志》2023年第1期1-7,共7页Journal of Oral and Maxillofacial Surgery

基　　金：国家自然科学基金(82000995、81670962);科技部重点研发专项(2018YFE0202200)。

摘　　要：目的:探究甲氨蝶呤(methotrexate,MTX)对磨牙牙胚发育的影响。方法:体外于体式显微镜下分离出胚胎13.5 d(E13.5)小鼠下颌第一磨牙(first molar,M1)牙胚,并将其加入到新鲜的器官培养液中。将MTX溶解于二甲基亚砜,分别配制成0.1、1.0、10.0μmol/L MTX的器官培养液,作为实验组(MTX组);在培养液中加入等量的二甲基亚砜,作为对照组。隔日拍照观察牙胚变化的情况。对出生后2 d(P2)至出生后6天(P6)的新生鼠按1、3、5 mg/kg的剂量进行腹腔注射MTX(隔日注射),于第2.5、4、6、8天时取样,对下颌第三磨牙(third molar,M3)进行组织学分析。结果:体外培养9 d后,观察到1.0、10.0μmol/L MTX组牙胚均死亡;0.1μmol/L MTX组牙胚的宽度、总高度及牙尖高度均出现异常。体内实验观察到3、5 mg/kg MTX组的P8小鼠下颌M3较对照组形态异常;凋亡检测结果表明,3 mg/kg MTX组下颌M3在牙发育过程中,其凋亡细胞较对照组多;免疫荧光结果表明,在P2.5、P4小鼠下颌M3中,3 mg/kg MTX组增殖细胞较对照组少,P6、P8小鼠下颌M3中,3 mg/kg MTX组增殖细胞较对照组多。结论:MTX会导致磨牙形态发育异常,出现牙胚体积减小、发育延缓等,这可能是因为牙发育过程中,MTX促进了牙胚细胞凋亡,在蕾状期及帽状期抑制了牙胚细胞增殖。Objective:To explore the effect of methotrexate(MTX)on the development of mouse molar tooth germ.Methods:In vitro,mandibular first molar(M1)tooth germs from the 13.5th day embryonic(E13.5)mouse were dissected under stereomicroscope and then cultured with fresh organ culture solution.In the experimental group,MTX was dissolved into the dimethyl sulfoxide(DMSO)to prepare organ culture solution containing 0.1,1.0,10.0μmol/L MTX;in the control group,the same volume of DMSO was added to the organ culture solution.And the change of germs was taken photos every other day.In vivo,1,3,5 mg/kg MTX solution was managed intraperitoneally in postnatal mouse every other day from the second postnatal day(P2)to P6,and the mandibular third molars(M3)were histologically evaluated at P2.5,P4,P6,P8.Result:After 9-day in vitro culture,it was observed that tooth germs were all dead in 1.0μmol/L MTX and 10.0μmol/L MTX group,and the tooth germs of 0.1μmol/L MTX showed abnormality in the width,total height and cusp height.In vivo,the morphology of mandibular M3 in 3 mg/kg MTX and 5 mg/kg MTX group showed more abnormalities than that in the control group.Apoptosis assay results showed that more apoptotic cells in 3 mg/kg MTX group than that in the control group.Immunofluorescent assay results showed the proliferative cells of mandibular M3 from P2.5 and P4 mouse in 3 mg/kg MTX group were less than that in the control group and proliferative cells from P6 and P8 in 3 mg/kg MTX group were more than that in the control group.Conclusion:MTX may cause the abnormalities in morphology development of teeth,with decreased volume of tooth germs and delayed development,which may be attributed to MTX promot-ing tooth germ cell apoptosis during the process of tooth development and inhibiting tooth germ cell proliferation at bud stage and cap stage.

关 键 词：甲氨蝶呤 牙发育 细胞增殖 细胞凋亡 

分 类 号：R782[医药卫生—口腔医学]