孕期尼古丁暴露对子代小鼠牙釉质形成的影响  

Effects of prenatal nicotine exposure on enamel formation of offspring mice

作　　者：田青鹭 赵迪芳 关淑元 周媛 周昕[1] 郑黎薇[1] Tian Qinglu;Zhao Difang;Guan Shuyuan;Zhou Yuan;Zhou Xin;Zheng Liwei(Department of Pediatric Dentistry,West China Hospital of Stomatology,Sichuan University&State Key Laboratory of Oral Diseases&National Clinical Research Center for Oral Diseases,Chengdu 610041,China;Department of Stomatology,Peking Union Medical College of Hospital,Peking Union Medical College,Chinese Academy of Medical Sciences,Beijing 100010,China)

机构地区：[1]四川大学华西口腔医院儿童口腔科、口腔疾病研究国家重点实验室、国家口腔疾病临床医学研究中心,成都610041 [2]中国医学科学院、北京协和医学院、北京协和医院口腔科,北京100010

出　　处：《中华口腔医学杂志》2023年第1期40-49,共10页Chinese Journal of Stomatology

基　　金：国家自然科学基金(82170921)。

摘　　要：目的研究孕期尼古丁暴露对子代小鼠牙釉质形成的影响及其可能的表观遗传学机制。方法通过随机数字表法将10只C57BL/6孕鼠分为对照组(皮下注射生理盐水)及孕期尼古丁暴露(prenatal nicotine exposure, PNE)组(皮下注射尼古丁溶液), 每组5只, 孕鼠生产后分别收集出生后0 d(postnatal day 0, P0;P14、P25含义依此类推)、P14、P25新生小鼠, 根据母代分组分为子代对照组及子代PNE组, 并记录P0、P25子代小鼠体质量。采用显微CT(micro-CT)扫描分析、扫描电镜和维氏显微硬度检测对子代小鼠牙槽骨和下颌切牙进行硬组织相关参数分析。提取P25子代小鼠下颌骨组织及第3代牙上皮干细胞(dental epithelial stem cell, DESC)中的总RNA, 通过实时荧光定量PCR检测成骨向及成釉向分化相关基因、增殖相关标志物和干细胞标志物的表达水平。采用石蜡切片免疫组织化学染色观察增殖细胞核抗原(proliferating cell nuclear antigen, Pcna)、釉原蛋白(amelogenin, Amelx)、甲基转移酶ZESTE增强子同源物2(enhancer of zeste homolog 2, Ezh2)、组蛋白H3第27位赖氨酸的三甲基化修饰(histone H3 trimethylated at lysine 27, H3K27me3)阳性染色分布及水平。使用细胞增殖(cell counting kit-8, CCK-8)试剂盒对外源性添加不同浓度(0.01、0.1、1 mmol/L)尼古丁以及Ezh2抑制剂GSK126的DESC进行细胞活力检测。结果 PNE组孕鼠较对照组更容易出现不良妊娠结局, P0时子代PNE组小鼠体质量[(0.99±0.02)g]显著低于子代对照组[(1.20±0.04)g](P<0.001);P25时, 子代PNE组小鼠体质量[(9.65±1.32)g]显著低于子代对照组[(15.26±1.70)g](P<0.001), 死产率[(46.40±9.30)%]显著高于对照组(0)(P<0.001)。P14和P25时, 子代PNE组小鼠下颌切牙釉质矿化沉积点与第一磨牙近中面的距离数值[分别为(-1 349±45)、(-506±380)μm]均较子代对照组[分别为(-1 192±147)、(504±198)μm]显著减小(P<0.05, P<0.001), 提示矿化沉积点延迟。子�Objective To investigate the effects of nicotine on the morphology,structure of offspring′s dental germ,enamel organ and other dental tissues and the further potential epigenetic mechanisms by establishing prenatal nicotine exposure mouse model.Methods Ten C57BL/6 pregnant mice were randomly divided into control group(physiological saline subcutaneous injection)and prenatal nicotine exposure(PNE)group(nicotine subcutaneous injection)by using a random number table.Postnatal day 0(P0),postnatal day 14(P14)and postnatal day 25(P25)offspring mice were collected for subsequent experiments.The offspring mice were divided into offspring control group and offspring PNE group according to the maternal group respectively.Weights of P0 and P25 offspring mice were recorded.Micro-CT,scanning electron microscope(SEM)and Vickers hardness test were performed to analyze the related parameters of hard tissues including alveolar bones and mandibular incisors.Total RNAs were extracted from mandible tissues and the third generation of dental epithelial stem cells(DESC)in P25 mice.The relative expression levels of osteogenic and ameloblastic differentiation related genes were measured by real-time quantitative PCR(RT-qPCR).Immunohistochemical stainings of paraffin sections were then performed to observe the distribution and expression level of proliferating cell nuclear antigen(Pcna),amelogenin(Amelx),histone H3 trimethylated at lysine 27(H3K27me3)and enhancer of zeste homolog 2(Ezh2).Cell counting kit-8(CCK-8)assays were used to detect the cell viabilities of DESCs after administrations of different concentrations of nicotine(0.01,0.1,1 mmol/L)and GSK126(an inhibitor of histone methyltransferase Ezh2).Results Compared with the control group,pregnant mice in PNE group were more likely to have adverse pregnancy outcomes,such as significantly lower offspring body weight[P0:offspring control(1.20±0.04)g,offspring PNE(0.99±0.02)g,P<0.001;P25:offspring control(15.26±1.70)g,offspring PNE(9.65±1.32)g,P<0.001]and increased stillbirths r

关 键 词：尼古丁 牙釉质 表观遗传学 牙上皮干细胞 牙釉质发育缺陷 

分 类 号：R715.3[医药卫生—妇产科学]