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  

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

作　　者：刘鑫[1] 黄兴[1] 徐智云[1] 杨德琴[1] 

机构地区：[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

出　　处：《华西口腔医学杂志》2016年第2期115-120,共6页West China Journal of Stomatology

基　　金：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)~~

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m RNA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。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 dlx2 a, dlx2 b, 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 m RNA probes successfully. Compared with DMSO control group, a low concentration（1×10-7 mol·L-1） of RA could up-regulate the expression of m RNA（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 dlx2 a were suppressed, and the appearance of dlx2 b 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.

关 键 词：视黄酸信号通路 牙齿发育 神经嵴 斑马鱼 

分 类 号：Q344[生物学—遗传学]