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  

The effect of pitx2 over expression on the expression of the genes involved in tooth development in the early stage

作　　者：陆琼琼 王娟[2] 赵庆顺[3] 李谨[2] 

机构地区：[1]杭州口腔医院,浙江杭州310000 [2]南京医科大学口腔医学研究所,江苏南京210029 [3]南京大学模式动物研究所,江苏南京210061

出　　处：《牙体牙髓牙周病学杂志》2013年第1期6-11,共6页Chinese Journal of Conservative Dentistry

基　　金：江苏省科技厅自然科学基金-面上项目(BK2010528);江苏高校优势学科建设工程资助项目(PAPD)

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。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 groupe I pitx2 mRNA at 200 pg/nL was microinjected into a ze- brafish embryo and in group Ⅱ at 100pg/nL; in group Ⅲ 100 pg/nL yellow fluorescent protein （YFP） mRNA was giv- en as the control and in group Ⅳ the natural mating was served as the wild type control. The development of the ze- brafish embryos was observed for 72 h. bmp4, fgf8, dlx2b, runx2a and lefl 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 val- ue analysis. RESULTS ： pitx2 mRNA was synthesized and identified. In group 1 66% embryos died in 24h after mi- croinjection, the development of the rest zebrafish embrvos was arrested or in dvsgenesis. The survival ratios of the oth-er 3 groupes were 84% or more. In groupe Ⅱ development arrest, pericardial edema, spinal deformity and eye malfor- mations were observed in 72 h ; bmp4 expression was down regulated, fgf8 , lef7 and mnx2a expressions were up reg- ulated, dlx2b expression was not changed. CONCLUSION： pitx2 over expression may impact the expression of the genes involved in tooth development in the early stage.

关 键 词：斑马鱼 PITX2 显微注射 半定量RT-PCR 

分 类 号：R780.2[医药卫生—口腔医学]