Pitx2基因功能下调对斑马鱼牙齿早期发育相关基因的影响

›› 2013, Vol. 33 ›› Issue (2): 78-83.

Pitx2基因功能下调对斑马鱼牙齿早期发育相关基因的影响

耿楠¹,张堃²,李谨¹

1. 南京医科大学口腔医学院
2. 南京医科大学口腔医院

收稿日期:2012-08-01 修回日期:2012-11-13 出版日期:2013-02-08 发布日期:2013-02-22
通讯作者: 耿楠 E-mail:gengnan1673@sina.com
基金资助:
江苏省科技厅自然科学基金

Effect of Pitx2 knock down on the early dental development related genes in Zebrafish

Received:2012-08-01 Revised:2012-11-13 Online:2013-02-08 Published:2013-02-22

摘要/Abstract

摘要： 目的通过Pitx2-morpholino反义寡核苷酸显微注射方法沉默斑马鱼Pitx2基因，研究Pitx2基因早期功能下调对斑马鱼牙齿发育相关的基因的影响。方法通过Morpholino反义寡核苷酸显微注射方法功能下调斑马鱼Pitx2基因，观察胚胎在72 h内的发育情况。并利用RT—PCR技术直接克隆Bmp4、Fgf8特异性基因片段，制作针对Bmp4、Fgf8的基因探针，对斑马鱼发育早期多个时段的胚胎进行整体原位杂交，观察Pitx2基因下调对Bmp4、Fgf8在斑马鱼腮弓表达的影响。结果成功合成Bmp4、Fgf8基因探针。显微镜下观察实验斑马鱼胚胎在72 h内的生长发育情况，发现经Morpholino显微注射斑马鱼胚胎出现发育迟缓或停止、心包水肿、脊索畸形、眼睛发育异常等形态学改变。原位杂交发现相对于野生型，Morpholino显微注射斑马鱼腮弓处Bmp4基因表达有所增强，Fgf8基因表达有所减弱。结论了解Pitx2基因的表达下调对斑马鱼早期发育的影响及牙齿发育相关基因Bmp4、Fgf8表达的影响。

关键词: 斑马鱼, pitx2, Morpholino, 原位杂交, zebrafish, Pitx2, Morpholino, In situ hybridization

Abstract: Objective The aim of this study was silence Pitx2 gene by Pitx2- morpholino oligos microinjection so as to explore the effect of down-regulated Pitx2 on tooth development related genes in zebrafish. Methods Pitx2 gene was down-regulated by morpholino oligos microinjection. We observed the development of the embryos in 72 hours with microscopes, synthesized gene probes for Bmp4、Fgf8 by cloning specific gene fragment of Bmp4 and Fgf8 with RT-PCR directly，and then carried out the in situ hybridization with embryos from different stages during early dental development to observe the effect of down-regulated Pitx2 on expression of Bmp4 and Fgf8 in zebrafish branchial arch in early dental development. Results We successfully synthesized gene probes for Bmp4 and Fgf8，and observed the development of the embryos in 72 hours with microscopes. Growth retardation or termination,. pericardial edema, spinal and eyes deformity appeared in the zebrafish embryos in Morpholino groups after Pitx2- morpholino oligos microinjection. In situ hybridization showed that down-regulated Pitx2 increased the expression of Bmp4, and repressed the expression of Fgf8 in branchial arch of zerafish after Pitx2- morpholino oligos microinjection, compared with wild zebrafish. Conclusion We found out the Effect of down-regulated Pitx2 on the early dental development related genes Bmp4 and Fgf8 in zebrafish.

中图分类号:

R349.64

耿楠张堃李谨. Pitx2基因功能下调对斑马鱼牙齿早期发育相关基因的影响[J]. 口腔医学, 2013, 33(2): 78-83.

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