用Ddb1基因子宫特异性敲除小鼠模型研究Ddb1在小鼠早期妊娠中的作用及分子机制

Ddb1 (UV-damaged DNA binding protein 1), a linker protein of the Ddb1-Cul4 E3 ubiquitin ligase complex, recognizes damaged DNA and initiates the nucleotide excision repair (NER) process. Ddb1 also functions in diverse cellular processes including DNA replication and cell cycle regulation. However, to date, the function of Ddb1 in mouse uterus during pregnancy are still unknown. The aim of this project is to examine the uterine expression of Ddb1 during pregnancy in mice and its regulation under pseudopregnancy, delayed implantation, steroid hormone treatment, and artificial decidualization conditions. Meanwhile, using the mice with conditional deletion of Ddb1 gene in uterus, we will study the function of Ddb1 during mouse different pregnancy stages. Our primary results showed that Ddb1 deletion mice are infertile, and uterine cell proliferation, apoptosis and differentiation are affected. We will explore in detail the mechanism by which Ddb1 regulates uterine cell proliferation, apoptosis and differentiation during different pregnancy stages.We will also identify molecules that can directly interact with Ddb1 and downstream signaling pathways affected by Ddb1 deletion in mouse uterus. Finally, we will try to rescue the reproductive defects caused by Ddb1 gene deletion by using genetic and pharmacological approaches. The proposed research will not only provide valuable information for understanding the molecular mechanism of Ddb1 in keeping normal function of uterus, but also provide new idea for embryo implantation research and clinical infertility treatment.

Ddb1是Ddb1-Cul4 E3 泛素连接酶的组成成分之一,识别损伤的DNA,启动核苷酸切除修复。Ddb1也通过泛素化其它蛋白参与DNA复制与细胞周期调控等生物学过程。目前，关于Ddb1在哺乳动物妊娠过程中的作用未见报道。本课题将利用小鼠早期妊娠、假孕、延迟着床、人工蜕膜化和类固醇激素处理等动物模型，阐明Ddb1在小鼠妊娠过程中的表达调控规律。并利用Ddb1基因子宫特异性敲除小鼠研究Ddb1在妊娠不同阶段的作用。初步结果表明Ddb1敲除小鼠不育，子宫细胞增殖、凋亡及分化受到影响。我们拟详细研究Ddb1在妊娠不同阶段对子宫细胞增殖、凋亡及分化的影响，鉴定Ddb1在子宫中的直接相互作用分子及受Ddb1影响的下游信号通路，并运用多种手段挽救Ddb1基因敲除鼠生殖缺陷。本研究将为阐明Ddb1维持子宫正常生理功能的分子机理提供重要依据，为胚胎着床研究和临床不孕不育治疗提供理论基础和新思路。

Ddb1是Ddb1-Cul4 E3泛素连接酶的组成成分之一，识别损伤的DNA，启动核苷酸切除修复。 Ddb1通过底物受体 DCAFs （DDB1-Cul4-associated factors）特异性地泛素化其它蛋白，参与 DNA 复制与细胞周期调控等生物学过程。目前，关于 Ddb1在哺乳动物妊娠过程中的作用未见报道。本课题利用小鼠早期妊娠、假孕、延迟着床、人工蜕膜化和类固醇激素处理等动物模型，发现Ddb1 在小鼠早期妊娠过程中的子宫腔上皮和腺上皮中有规律表达，并受雌激素和激活胚胎的调控。Cul4在小鼠早期妊娠过程中的表达模式与Ddb1类似，接头蛋白VPRBP、Dtl和Ddb2等也在小鼠早期妊娠不同阶段表达，表明可能在小鼠早期妊娠不同阶段存在不同的Cul4-Ddb1-DCAFs复合体发挥不同作用。子宫特异性Ddb1敲除小鼠不育，胚胎在输卵管中的运输受阻，子宫接受态破坏，蜕膜化削弱。子宫特异性Ddb1敲除导致子宫细胞增殖能力降低、凋亡增加、蜕膜化相关基因Hoxa10和CyclinD3等表达减弱。利用转录组测序技术我们发现与野生型相比DDB1子宫特异性敲除的蜕膜组织有445个基因表达上调，326个基因表达下调。本研究为阐明 Ddb1 维持子宫正常生理功能的分子机理提供了重要依据，为胚胎着床研究和临床不孕不育治疗提供理论基础和新思路。