华西口腔医学杂志

• 基础研究 • 上一篇    下一篇

视黄酸信号通路调控咽弓神经嵴影响斑马鱼牙齿发育的研究

刘鑫 黄兴 徐智云 杨德琴   

  1. 重庆医科大学附属口腔医院牙体牙髓病科·口腔疾病与生物医学重庆市重点实验室,重庆 401147
  • 收稿日期:2015-08-12 修回日期:2015-10-16 出版日期:2016-04-01 发布日期:2016-04-01
  • 通讯作者: 杨德琴,教授,博士,E-mail:yangdeqin@gmail.com
  • 作者简介:刘鑫,住院医师,硕士,E-mail:tijiuwei@outlook.com
  • 基金资助:

    国家自然科学基金(31571508,31371473);重庆市第七批重点学科建设项目《牙体牙髓病学》(2011)

Retinoic acid signal pathway regulation of zebra fish tooth development through manipulation of the differentiation of neural crest

Liu Xin, Huang Xing, Xu Zhiyun, Yang Deqin.   

  1. Dept. of Conservative Dentistry and Endodontics, Stomatological Hospital of Chongqing Medical University, Chongqing Key Laboratory of Oral Diseases and Biomedical Sciences, Chongqing 401147, China
  • Received:2015-08-12 Revised:2015-10-16 Online:2016-04-01 Published:2016-04-01
  • Contact: Yang Deqin, E-mail: yangdeqin@gmail.com.
  • Supported by:

    Natural Science Foundation of China (31571508, 31371473); The Seventh Batch of Key Discipline Construction Project of Chongqing—Endodontics and Operative Dentistry (2011).

摘要:

目的 研究视黄酸(RA)信号通路在斑马鱼颅神经嵴迁移至咽弓神经嵴对牙齿发育的影响。方法 将野生型斑马鱼胚胎与转基因斑马鱼胚胎各分为3组,分别采用1×10-7~6×10-7 mol·L-1梯度浓度的RA(RA处理组)、1×10-7 mol·L-1的4-二乙氨基苯甲醛(DEAB)(DEAB处理组)、与RA处理组相应浓度的二甲基亚砜(DMSO对照组)处理24 hpf胚胎9 h。制备dlx2a、barx1、dlx2b基因的反义探针,运用整胚原位杂交技术检测野生型斑马鱼48~72 hpf胚胎的dlx2a、dlx2b、barx1基因表达,荧光显微镜观察转基因斑马鱼4 dpf胚胎。结果 获得3个基因的反义mRNA探针。与DMSO对照组相比,1×10-7 mol·L-1RA处理组barx1、dlx2a在咽弓神经嵴的表达明显增强,并有由咽弓神经嵴向后段咽弓牙齿萌出位点迁移的趋势,绿色荧光向周边咽弓扩散;4×10-7 mol·L-1 RA处理组胚胎致畸率和死亡率极高,3×10-7 mol·L-1 RA处理组1/3胚胎发育迟缓。DEAB处理组神经嵴发育不良,barx1、dlx2a表达降低,dlx2b在牙齿位点的表达有所延迟。结论  RA能够通过调控咽弓神经嵴发育过程,进而调控牙齿发育前体细胞,最终达到调控牙齿发育的过程。

关键词: 视黄酸信号通路, 牙齿发育, 神经嵴, 斑马鱼

Abstract:

Objective  To investigate the mechanism of retinoic acid (RA) signal in dental evolution, RA is used to explore the influence of the mechanism on neural crest’s migration during the early stage of zebra fish embryos. Methods We divided embryos of wild type and transgenic line zebra fish into three groups. 1×10-7 to 6×10-7 mol·L-1 RA and 1×10-7 mol·L-1 4-diethylaminobenzaldehyde (DEAB) were added into egg water at 24 hpf for 9 h. Dimethyl sulfoxid (DMSO) with the concentration was used as control group. Then, antisense probes of dlx2a, dlx2b, and barx1 were formulated to perform whole-mount in situ hybridization to check the expressions of the genes in 48 hpf to 72 hpf embryos. We observed fluorescence of transgenic line in 4 dpf embryos. Results We obtained three mRNA probes successfully. Compared with DMSO control group, a low concentration (1×10-7 mol·L-1) of RA could up-regulate the expression of mRNA (barx1, dlx2a) in neural crest. Obvious migration trend was observed toward the pharyngeal arch in which teeth adhered. Transgenic fish had spreading fluorescence tendency in pharyngeal arch. However, a high concentration (4×10-7 mol·L-1) of RA malformed the embryos and killed them after treatment. One third of the embryos of middle concentration (3×10-7 mol·L-1) exhibited delayed development. DEAB resulted in neural crest dysplasia. The expression of barx1 and dlx2a were suppressed, and the appearance of dlx2b in tooth was delayed. Conclusion RA signal pathway can regulate the progenitors of tooth by controlling the growth of the neural crest and manipulating tooth development.

Key words: retinoic acid signal pathway, tooth development, neural crest, zebrafish