国际口腔医学杂志

国际口腔医学杂志 ›› 2022, Vol. 49 ›› Issue (3): 317-323.doi: 10.7518/gjkq.2022048

• 论著 • 上一篇    下一篇

2,3,7,8-四氯二苯二噁英诱导C57BL小鼠腭裂发病机制的研究

罗枭(),蔡生青,石冰,李承浩()   

  1. 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四川大学华西口腔医院唇腭裂外科 成都 610041
  • 收稿日期:2021-08-12 修回日期:2021-12-11 出版日期:2022-05-01 发布日期:2022-05-09
  • 通讯作者: 李承浩
  • 作者简介:罗枭，硕士，Email：2577543004@qq.com
  • 基金资助:
    四川省科技厅重点研发计划(2019ZDYF1658)

Investigation of the mechanism of 2,3,7,8-tetrachlorodiphenyl dioxin-induced cleft palate mice model

Luo Xiao(),Cai Shengqing,Shi Bing,Li Chenghao.()   

  1. State Key Laboratory of Oral Diseases＆National Clinical Research Center for Oral Diseases＆Dept. of Cleft Lip and Palate Surgery, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China
  • Received:2021-08-12 Revised:2021-12-11 Online:2022-05-01 Published:2022-05-09
  • Contact: Chenghao. Li
  • Supported by:
    Key Project of Science and Technology Department in Sichuan Province(2019ZDYF1658)

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摘要： 目的

探究2，3，7，8-四氯二苯二噁英（TCDD）诱导的腭裂模型中对中嵴上皮细胞影响的机制。

方法

E12.5孕鼠随机分为对照组和实验组，实验组孕鼠胃饲质量浓度为4 μg·mL-1的TCDD；对照组孕鼠胃饲蓖麻油。于E18.5在体视显微镜下检测各组腭发育情况；分别在E13.5、E14.5、E15.5取胎鼠腭部组织行苏木精-伊红（HE）染色和免疫组织化学染色，观察小鼠腭部形态和蛋白酶活化受体/非典型蛋白激酶C（PAR/aPKC）复合物、β-连环蛋白的表达情况；用逆转录聚合酶链反应（RT-PCR）定量检测mRNA表达；Western blot定量检测PAR/aPKC复合物。

结果

TCDD组胎鼠在E18.5全部发生腭裂，对照组未见腭裂产生。RT-PCR定量检测，PAR/aPKC复合体mRNA在E13.5表达最强，E14.5、E15.5的表达减弱。β-连环蛋白在E14.5表达最强，E13.5次之，E15.5表达最弱；E13.5、E14.5 TCDD组β-连环蛋白的表达明显低于对照组，E15.5高于对照组（P<0.01）。Western blot检测，PAR/aPKC复合体的表达随着腭发育而降低。免疫组织化学染色显示β-连环蛋白在对照组E13.5、E14.5腭中嵴上皮细胞内呈强阳性表达。

结论

TCDD有可能通过干扰PAR/aPKC复合体协同β-连环蛋白参与诱导小鼠腭上皮融合异常，而导致腭裂。

关键词: 蛋白酶活化受体/非典型蛋白激酶C复合体, 细胞极性, 四氯二苯二噁英, 腭裂, β-连环蛋白

Abstract: Objective

This study aims to investigate the mechanism in which 2,3,7,8-tetrachlorodiphenyl dioxin (TCDD) induces palatal cleft in C57BL mice by affecting palatal ridge epithelial cells.

Methods

C57BL mice were divided into the control and experimental group. The pregnant mice in the experimental group were treated with TCDD at embryonic day 12.5 (E12.5), while the mice in the control group were treated with an equivalent volume of castor oil. Haematoxylin and eosin (HE) staining, real-time quantitative polymerase chain reaction, Western blot and immunohistochemistry were employed to determine the expression level of protease-activated receptor/atypical protein kinase C (PAR/aPKC) complex and β-catenin in foetal mouse palatal processes in the phase of the control and experimental group.

Results

Cleft palate was induced in all foetal mice in TCDD group at E18.5 but not in the control group. The reverse transcription-polymerase chain reaction (RT-PCR) quantitative results show that the expression of PAR/aPKC complex mRNA was the strongest at E13.5 but weakened at E14.5 and E15.5. The expression level of β-catenin was the highest at E14.5, followed by E13.5, and the lowest at E15.5. The expression of β-catenin in TCDD group at E13.5 and E14.5 was significantly lower than that of the control group but higher at E15.5 (P<0.01). Western blot analysis revealed that the expression of PAR/aPKC complex decreased with palatal development. Strong positive expression of β-catenin in the palatal ridge epithelial cells was observed at E13.5 and E14.5 in the control group by immunohistochemical staining.

Conclusion

TCDD may induce failure of palatal epithelial fusion in mice by interfering with the expression of PAR/aPKC complex and β-catenin, resulting in cleft palate.

Key words: protease-activated receptor/atypical protein kinase C complex, cell polarity, tetrachlorodiphenyl dioxin, cleft palate, β-catenin

中图分类号: 

  • R 34

表　1

引物序列"

基因上游引物（5’—3’）下游引物（5’—3’）
PAR3CATAGTGCTCACGCCTCAACCGTGATGCTGAATCCCAGTCCTT
PAR6GAGTTTTACGGACTGCTGCAAGCTTCTTCCGGTGGTTGTCG
aPKCACGACCAGATTTACGCCATGAGAAGCAGGAGTGTAAGCCAACC
β-连环蛋白AAGGAAGCTTCCAGACATGCAGCTTGCTCTCTTGATTGCC
GAPDHCCTGTTCGACAGTCAGCCGCGACCAAATCCGTTGACTCC

图1

对照组及TCDD组C57BL/6J小鼠腭胚突发育过程 HE × 10A~C依次为：对照组E13.5、E14.5、E15.5；D~F依次为：实验组E13.5、E14.5、E15.5；P： 腭板，T：舌体；→：融合。"

图2

RT-PCR检测PAR/aPKC复合体mRNA基因的表达**：P<0.01；***：P<0.001。"

图3

Western blot检测PAR/aPKC复合体在蛋白水平的表达**：P<0.01；***：P<0.001。"

图4

β-连环蛋白在对照组和TCDD组腭中嵴上皮中的表达 SABC × 40A~C依次为：对照组E13.5、E14.5、E15.5；D~F依次为：实验组E13.5、E14.5、E15.5；P： 腭板，T：舌体；→：融合。"

图5

RT-PCR检测β-连环蛋白在腭发育不同时间点的表达**：P<0.01；***：P<0.001。"

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