【摘要】 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 pg/nL;实验2组注射pitx2 mRNA 100 pg/nL;实验3组注射YFP mRNA 100 pg/nL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 pg/nL 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。更多还原

【Abstract】 AIM: To study the effect of pitx2 over expression on the expression of the genes involved in tooth 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(0 h) were used in each of the following groupes: in groupeⅠ pitx2 mRNA at 200 pg/nL was microinjected into a zebrafish embryo and in group Ⅱ at 100pg/nL;in group Ⅲ100 pg/nL 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 lef1 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 groupⅠ66% 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 Ⅱ 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.更多还原