XBP1调控Nkx2.5影响心脏发育的机制研究

The cascade activation of the cardiogenic transcription factors orchestrates the early steps in heart formation. And any error in the regulation causes congenital heart disease(CHD). So far the networks of interacting transcription factors are only partially understood, and it is important to search for novel transcription factor and clarify the interactions for deciphering the cardiogenesis. XBP1 acts as a transcription factor. It was reported that XBP1 homozygous mutant mice have an embryonically lethal phenotype associated with heart dysfunction, causing them to die between embryonic day 10.5 (E10.5) and E14.5 of gestation which indicates that XBP1 plays an essential role in cardiogenesis, but it is unknown if it is involved in the transcription factors networks. Bioinformatics assay shows that the upstream sequence of Nkx2.5 contains a putative ER stress element (ERSE) where XBP1 may bind, which indicates that XBP1 may involve in the transcriptional regulation of Nkx2.5 which is one of the earliest expressed key transcription factor. Our data showed that knockdown of XBP1 in xenopus lavies caused heart defects with reduced expression of Nkx2.5. So we presume that XBP1 may regulate Nkx2.5 at transcription level during heart development. In order to validate the hypothesis, we will use gain-of-function, loss-of-function and rescue assay to test the effects of XBP1 on heart development and the correlation of XBP1 and Nkx2.5.Then we will detect the transcriptional regulation of XBP1 on Nkx2.5. Our study will contribute to understand the mechanism of initiation of heart development,CHD and new approaches to the treatment.

心脏发育过程中特异性转录因子的级联激活起重要调控作用，调控异常可导致先天性心脏病。目前对转录因子调控网络认识有限，而发现新的转录因子，了解其相互作用是心脏发育研究的重要内容之一。转录因子XBP1功能缺失导致小鼠死于心脏发育异常，提示其在心脏发育中有重要作用，但是否参与转录因子调控网络尚不清楚。生物信息学分析发现Nkx2.5调控区存在XBP1结合位点，提示其可能参与Nkx2.5的转录调控。前期研究发现封闭XBP1导致非洲爪蟾心脏发育不全，且Nkx2.5表达下降。我们认为XBP1 可能通过转录调控Nkx2.5而影响心脏发育。为验证此假说，本课题拟在非洲爪蟾胚胎中利用过表达、敲降及补救方法，验证XBP1对心脏发育的影响及与Nkx2.5的相关性，进而验证XBP1对Nkx2.5的转录调控作用。本研究不仅有助于理解心脏早期发育机制，也有助于认识先天性心脏病发病机制、探寻新的干预方法。

心脏发育过程中特异性转录因子的级联激活起重要调控作用，调控异常可导致先天性心脏病。目前对转录因子调控网络认识有限，发现新的转录因子，了解其相互作用是心脏发育研究的重要内容之一。转录因子XBP1功能缺失导致小鼠死于心脏发育异常，提示其在心脏发育中有重要作用，但XBP1是否参与转录因子调控网络尚不清楚。本研究利用非洲爪蟾作为模式动物，研究XBP1在心脏发育中的作用及机制。首先利用原位杂交、Western blot等方法检测了XBP1的表达模式，发现XBP1表达起始于受精卵期，随后持续表达于胚胎发育各阶段，在尾芽期XBP1表达于多种组织，尤其在心脏组织高表达；进而利用基因过表达及敲降方法研究XBP1对心脏发育的影响，发现XBP1抑制心脏发育，影响心脏发育早期标志基因的表达；进一步利用补救实验、免疫荧光双染、染色质免疫共沉淀方法发现，XBP1可通过转录调控Nkx2.5.而发挥作用；此外，利用TUNNEL及免疫组化方法发现敲降XBP1后凋亡细胞数增加而增殖细胞数减少。本研究结果提示XBP1参与心脏发育的转录因子调控网络，为更好地理解心脏早期发育、先天性心脏病的发生、心肌细胞的再生机制提供线索。