pitx2基因早期过量表达对斑马鱼牙齿生长发育相关基因的影响

摘要

AIM: To study the effect of pitx2 over expression on the expression of the genes involved inrntooth development in the early stage. METHODS: Specific gene fragment of pitx2 gene was cloned and the restriction enzyme cutting site in the two terminals was inserted. Pitx2 mRNA was synthesized by reverse transcription -polymerase chain reaction( RT-PCR) using the pitx2 coding sequence( CDS) as the template. 100 zebrafish embryos (0h) were used in each of the following groupes; in groupe Ⅰ pitx2 mRNA at 200 pgL was microinjected into a zebrafish embryo and in group Ⅱ at 100pgL; in group Ⅲ100 pgL yellow fluorescent protein ( YFP) mRNA was given as the control and in group Ⅳ the natural mating was served as the wild type control. The development of the zebrafish embryos was observed for 72 h. Bmp4 , fgf8 , dlx2b , runx2a and left expressions of the embryos in the 4 groups were examined at 24 h,48 h and 72 h respectively after microinjection by semi-quantitative RT-PCR and grayscale value analysis. RESULTS: pitx2 mRNA was synthesized and identified. In group Ⅰ 66% embryos died in 24h after microinjection , the development of the rest zebrafish embryos was arrested or in dysgenesis. The survival ratios of the other 3 groupes were 84% or more. In groupe fl development arrest, pericardial edema, spinal deformity and eye malformations were observed in 72 h; bmp4 expression was down regulated, fgf8 , lef1 and runx2a expressions were up regulated , dlx2b expression was not changed. CONCLUSION: pitx2 over expression may impact the expression of the genes involved in tooth development in the early stage.%目的:研究pitx2基因早期过量表达对斑马鱼牙齿发育相关基因的影响.方法:利用RT-PCR技术克隆pitx2特异性基因片段；插入特异性酶切位点后以之为模板体外合成pitx2 mRNA.选取正常交配获得的斑马鱼胚胎,随机分为4组采用显微注射技术构建pitx2过表达模型:实验1组注射pitx2 mRNA200 pgL；实验2组注射pitx2 mRNA 100 pgL；实验3组注射YFP mRNA 100 pgL作为对照；实验4组为不注射任何基因作为野生型.分别于注射后24、48、72 h观察胚胎发育情况；利用RT-PCR检测5个与斑马鱼牙齿发育相关基因的表达情况.结果:成功克隆含有pitx2基因CDS的片段并体外合成mRNA.显微镜下观察发现:pitx2早期过量表达可造成胚胎发育停止、心包水肿、脊索变形和眼部发育畸形等形态学改变.相关基因检测显示,与对照组和野生型组相比,注射100 pgL pitx2 mRNA组bmp4表达明显下调(P＜0.05)；fgf8 、lef1、runx2a表达均明显上调(P ＜0.05)；dlx2b表达各组之间未见明显差异(P＜0.05).结论:pitx2早期过量表达对斑马鱼牙齿发育相关基因的表达有影响.