【作者】 孔凡涛；

【导师】 吴琼；

【作者基本信息】 哈尔滨工业大学 ， 生物化学与分子生物学， 2011， 硕士

【摘要】 小鼠Klhdc10基因是Kelch基序重复超家族成员之一。目前,研究发现该家族的很多基因参与多种生理过程并且在发育过程中对组织形态发生起重要作用。然而,尚未见关于Klhdc10的研究报道。为了探究Klhdc10在小鼠胚胎发育中的时空表达模式,本文通过半定量RT-PCR、原位杂交和实时荧光定量RT-PCR等技术,对Klhdc10基因在发育期的表达模式进行分析,以期为今后功能的研究奠定基础。生物信息学分析显示,小鼠Klhdc10与人源KLHDC10氨基酸序列的同源性达96.61%,并且都含有2个高度保守的Kelch基序。半定量RT-PCR结果显示,在胚胎发育的9.5-18.5天(E9.5-E18.5)的小鼠全胚胎中,均能检测到Klhdc10基因mRNA的表达,在E11.5天表达量最高。原位杂交结果显示,在E9.5天,Klhdc10基因表达信号主要集中在颅面部,特别是在端脑和视泡部位信号最强。在E10.5天和E11.5天,表达信号主要集中在端脑、间脑、中脑和菱脑。在E12.5和E15.5天小鼠的脑、舌及背部肌肉中均检测到较强的Klhdc10 mRNA信号,并且在E15.5天小鼠的肝和肾中也有较强表达。实时定量RT-PCR结果显示,Klhdc10的mRNA在E12.5、E15.5和E18.5天小鼠脑中有较高的表达趋势,而在心和肺中表达量相对较低;在E12.5、E15.5天的舌和肾及E18.5天的肝中也有较高的表达。进一步检测其在脑发育过程中的表达,结果显示Klhdc10在E12.5、E15.5和出生后第0天(P0)天小鼠脑的新脑皮、小脑和海马等部位广泛表达,然而在P7天主要在小脑有较强的表达。在脑中,Klhdc10的mRNA表达水平随着胚胎的发育逐渐升高,在E15.5天出现峰值,随后表达量虽有下降的趋势,但一直到出生仍维持相对较高的水平。在胎盘发育过程中,Klhdc10表达信号在E10.5和E12.5天主要集中在迷路层,而在E15.5、E16.5和E18.5天主要位于成胶质细胞层。Klhdc10的mRNA在E11.5-E15.5天胎盘中保持较低的表达水平,在E16.5天表达量出现最大值,随后表达量有下降的趋势但仍然保持相对较高的表达水平。以上研究结果显示Klhdc10在小鼠胚内和胚外胎组织中普遍表达并呈现动态的表达模式,暗示其可能参与胚内及胚外组织的发生过程,并且在小鼠的胚胎发育过程中可能起重要的作用。更多还原

【Abstract】 Mouse Klhdc10 gene belongs to a member of the Kelch domain containing proteins superfamily of which many members have been well studied. Many members are proved to participate in some physiological processes and are important for tissue morphogenesis during embryonic development. However, so far there are not any studies about Klhdc10. In this study, we characterized the expression of Klhdc10 by semiquantitative RT-PCR, in situ hybridization and real-time quantitative RT-PCR (qRT-PCR) in order to reveal Klhdc10 expression patterns during mouse embryonic development and make basis for further function study.Bioinformation suggested that the sequence similarity between mouse Klhdc10 and human KLHDC10 protein is 96.61%. Klhdc10 and KLHDC10 have two highly conserved Kelch motifs, respectively. Semi-quantity RT-PCR indicated that Klhdc10 is widely expressed from embryonic day 9.5 (E9.5) to E18.5 with the highest expression at E11.5. In situ hybridization indicated that Klhdc10 signals are prominent in craniofacial regions, especially in telencephalon and optic vesicle at E9.5. Klhdc10 signals were obviously detected in the telencephalon, diencephalon, mesencephalon and rhombencephalon at E10.5 and E11.5. Abundant signals were observed in brain, tongue and muscle of back at E12.5 and E15.5. Moreover, at E15.5, there are also stronger signals in liver and kidney. QRT-PCR results indicated that Klhdc10 mRNA was highly expressed in brain at E12.5, E15.5 and E18.5; while constantly lower in lung and heart. High expression levels were also detected in tongue and kidney (E12.5, E15.5) and liver (E18.5). Further studies suggested that Klhdc10 was widely expressed in the brain such as neocortex, cerebellum and hippocampus at E12.5, E15.5 and postnatal day 0 (P0); however, Klhdc10 signal was mainly confined to cerebellum at P7. Klhdc10 mRNA expression in the brain was increasing as development progressing and peaked at E15.5, while decreased thereafter but constantly maintaining relatively high expression until to birth.In placentas, Klhdc10 signals were abundantly detected in labyrinthine layer at E10.5 and E12.5, while mainly located in spongiotrophoblast layer at E15.5, E16.5 and E18.5. Klhdc10 mRNA was low from E11.5 to E15.5 and was highly expressed at E16.5, maintaining high expression level thereafter. Our results suggested that Klhdc10 was widely expressed in embryonic and extraembryonic tissues, exhibiting dynamic expression patterns. Klhdc10 is likely to participate in the morphogenesis of diverse embryonic and extraembryonic tissues, playing potential important roles in mouse embryonic development.更多还原