【作者】 杨柳；

【导师】 肖君华；

【作者基本信息】 东华大学 ， 生物化学与分子生物学， 2018， 硕士

【摘要】 哺乳动物的肢体发育涉及细胞增殖、分化、迁徙和凋亡,同源异形盒基因(HOX)、音猬因子(Sonic hedgehog,SHH)、成纤维生长因子(fibroblast growth factor,FGF)和WNT信号通路在其中发挥重要作用。肢体发育初期的肢芽主要由顶外胚层嵴(AER),渐进带(PZ)和极性活化区(ZPA)三部分组成。AER区是肢体生长的主要信号中心,表达一些FGF家族成员基因,如Fgf4和Fgf8。PZ区为AER内侧有着旺盛分裂能力的间质细胞区域,主要由Hoxa基因簇和Hoxd基因簇调控。ZPA区存在于肢芽的后侧,SHH和骨形成蛋白(bone morphogenetic protein,BMPs)等基因在其中起到表达调控作用。也有文献报道,肢体发育异常也会并发不孕不育症。由于本实验后肢性状有不完全的外显率,但母体相关的胚胎致死表型十分稳定,因此本实验以胚胎致死的表型为QTL的定位标准。首先,将性状小鼠与C57BL/6品系小鼠杂交,建立F2代同类系小鼠实验动物模型。在8号染色体上的相关区段,选择平均分布的4个SNP位点,再通过使用聚合酶链式反应-连接酶检测法(PCR-LDR)分型技术,对小鼠进行基因分型。通过对相关QTL精细定位后,本实验成功地将之前粗略定位的小鼠8号染色体上的10Mb缩短到2.5Mb(68.3-70.8Mb)。并通过MGI小鼠基因数据库上相关QTL区段的基因信息与小鼠二代测序信息比对,初步筛选出10个候选基因。接着,本实验为研究小鼠胚胎后肢发育阶段(E10.5,E11.5,E12.5,E13.5)相关基因(主要为Hox基因家族)与候选基因的共表达情况,挑选了一个候选基因Pbx4,与Hox基因家族、Wnt5a、Pitx1、Fgf8、Shh等43个与肢体发育相关的基因建立了表达谱,并建立了q PCR array技术手段,用于后续实验的大量候选基因研究工作。首先,选取了Hox基因家族、Wnt5a、Pitx1、Fgf8、Pbx4、Shh等44个与肢体发育相关的基因为检测基因,选取β-actin和Gapdh为看家基因,同时还设置了基因阴性对照组和样品阴性对照组,然后以加热烘干的方法将基因引物固定在PCR板上,加入样品和qPCR反应物,进行实时定量PCR反应。然后,对建立的qPCR array进行了评估工作,通过样品梯度稀释的方法检验了其扩增效率;统计所有表达谱基因的Ct值标准差,评估了该方案的重复性;通过qPCR array的溶解曲线和PCR产物的琼脂糖凝胶电泳证明了该技术手段特异性强的优点。通过已建立的qPCR array检测了C57BL/6(简称B6)品系小鼠胚胎后肢发育时期Hox家族、Wnt5a、Pitx1、Fgf8、Pbx4、Shh等基因的表达差异。以E10.5为对照,检测出在后肢发育时期基因有着两种共表达模式,即Hoxb6、Hoxb8、Hoxc8、Hoxc9、Hoxc10、Hoxd9、Shh、Hoxc9、Hoxc10、Hoxc11、Hoxd9、Hoxd12、Fgf8和Pitx1等基因的相对表达量呈先上调后下调的表达模式;Hoxa11、Hoxa13、Hoxc12、Hoxc13、Hoxd13等基因表达出现下调,候选基因Pbx4的相对表达水平与这种共表达模式相似。另外也有少部分Hox基因在小鼠后肢发育时期表达水平无明显变化。综上所述,本实验第一部分首先对肢体发育缺陷及胚胎致死的小鼠进行了染色体上相关QTL的精细定位,初步筛选了挑选候选基因。第二部分实验为研究小鼠胚胎时期后肢发育相关基因的表达状况,建立了高效稳定、特异性强且重复性好的qPCR array基因检测方案,检测结果描述了在C57BL/6品系小鼠胚胎肢体发育期Hox基因家族、Wnt5a、Pitx1、Fgf8、Pbx4、Shh等基因的相对表达差异,建立了后肢发育基因表达谱,为肢体发育研究工作开辟了新思路,也为之后的研究工作提供了小型数据库,对肢体发育过程中探索Hox基因表达调控网络具有重要意义。更多还原

【Abstract】 The development of the mammalian limbs involves the cellular proliferation,differentiation,migration and apoptosis.The genes of HOX,SHH(Sonic hedgehog),FGF(fibroblast growth factor)and WNT signaling pathways play an important role in development of the mammalian limbs.The limb buds are mainly composed of three parts: the apical ectodermal ridge(AER),the progressive zone(PZ),and the polar activation zone(ZPA).The region of ARE is the main signal center of the limbs growth,and expresses some members of the FGF genes family,such as Fgf4 and Fgf8.The region of PZ is a mesenchymal cell region with strong splitting ability in the inner part of AER.It is mainly controlled by Hoxa genes and Hoxd genes.The region of ZPA exists in the posterior part of limb buds,and the genes of SHH and BMPs play a regulatory role in it.It is reported in literature that the abnormal limbs development may also complicate the infertility.There is the incomplete penetrance in the hind limb traits,but the maternal lethal phenotype is very stable.And we used the embryonic lethal phenotypes for fine mapping.Firstly,the wild mice were hybridized with the C57BL/6 strains mice,and the F2 mice experimental animal models were established.In the relevant sections of the chromosome 8,the 4 SNPs loci were selected,and then the genotyping was carried out by using the PCR-LDR typing technique.By fine mapping of the related QTL,the experiment successfully shortened the 10 Mb to 2.5Mb on the chromosome 8.And 10 candidate genes were screened by comparing the genes information of QTL segments in the MGI databases with the sequencing information of the mice.In order to study the co-expression of related genes(Hox genes family)with the candidate genes(Pbx4)in the developmental stage of the mice embryonic hind limbs(E10.5,E11.5,E12.5,E13.5),the expression profiles and a q PCR array technique was established.These were used to detect and analyze a large number of candidate genes for subsequent experiments.First,the Hox genes family,Wnt5 a,Pitx1,Fgf8 and Shh were selected for the detection,and the beta-actin and Gapdh were selected as the housekeeping genes.At the same time,the negative gene controls and sample negative controls were also set up.The gene primers were fixed on the PCR plates by heating and drying,and then the samples and q PCR reactants were added to the real time quantitative PCR reactions.In order to evaluate the q PCR array,and the amplification efficiency was tested by the gradient dilution methods.The standard deviation of the Ct values of all expressed genes was counted,and the reproducibility of the scheme was evaluated.The advantages of this technique were proved by the dissolution curves of q PCR array and agarose gel electrophoresis of the PCR products.In this study,we established a real-time quantitative polymerasechain reaction array method to study the expression differences of the genes of Hox,Wnt5 a,Pitx1,Fgf8 and Shh in the development stages of the C57BL/6 mice embryonic hind limbs.Using E10.5 as a control,we detected two kinds of the genes co-expression patterns in the period of the development of the mice hind limbs.The expression of the Hoxb6,Hoxb8,Hoxc8,Hoxc9,Hoxc10,Hoxd9,Shh,Hoxc9,Hoxc10,Hoxc11,Hoxd9,Hoxd12,Fgf8 and Pitx1 genes were down-regulated after up-regulated.And the expression of Hoxa11,Hoxa13,Hoxc12,Hoxc13 and Hoxd13 were down-regulated,and the candidate gene of the Pbx4 had the similar expression patterns.In addition,a small number of the Hox genes did not change significantly during the development of the mice hind limbs.To sum up,in the first part of this experiment,the fine mapping of the chromosomal related QTL was carried out on mice with the limbs developmental defects and embryonic lethality,and the ranges of the candidate genes was shortened.The candidate genes were screened preliminarily.The second part of the experiment is to study the expression of the genes related to hind limbs development in mice embryos,and the experiment established a stable,highly specific and reproducible q PCR array gene detecting program.The detection results described the relative expression differences of the Hox genes family,Wnt5 a,Pitx1,Fgf8,Pbx4 and Shh in the C57BL/6 mice embryonic hind limbs development periods,and established the gene expression profiles of the hind limbs development.It opens up new ideas for the limbs development work,also provides a small database for the future research work.It is important to explore the regulation networks of the Hox genes expression during the limbs development.更多还原