叶酸缺乏通过mTOR诱发细胞自噬紊乱导致子宫内膜蜕膜化异常

The mechanism study on folate deficiency resulting in birth defects mainly forced on fetal development. It is very little to study it from the point of view in mother’s uterine. Our group have do some research around the effect of folate on endometrium and it was found that uterine receptivity was normal while decidualization was impaired in folate deficiency. However, it is not known how the decidualization was influenced in folate deficiency. Autophagy, which is mainly regulated by mTOR, is reported to play an important role on cell homeostasis. We have reported mTOR signaling was essential for normal endometrium function and in our pre-study, the autophagy related key molecules LC3, P62 and Atg5 were expressed abnormally in folate deficient endometrium. Considering all these, we speculate that folate deficiency would result in imbalanced autophagy though mTOR signaling and further impair endometrium decidualization, and cause adverse pregnancy outcomes at last. Impaired decidualization model in the condition of folate deficiency will be established in vivo and in vitro and a serious of molecular biological technique will be used to study this hypothesis around all steps in autophagy. By this study, it will be helpful to explain the mechanism of birth defects induced by folate deficiency and to provide new clue for birth defects prevention.

目前叶酸缺乏导致胎儿出生缺陷的机理探索主要集中在胎儿自身发育方面，从孕早期母体的角度探寻其发生机制鲜有报道。课题组已发现叶酸缺乏虽不影响子宫内膜容受性的建立，但着床后的子宫内膜蜕膜化进程显著受损，其机制尚不明晰。自噬是生物体内一种相对保守的对物质进行分解代谢的过程，参与维持细胞内环境稳定，主要受到mTOR信号通路调节。课题组曾报道mTOR可参与调节子宫内膜正常功能，前期研究亦确证叶酸缺乏孕鼠蜕膜组织中自噬相关分子LC3、P62、Atg5表达异常，故提出叶酸缺乏可能通过mTOR信号通路引发细胞自噬紊乱干扰母体子宫内膜蜕膜化正常进程进而造成不良妊娠结局的科学假设。本项目将建立叶酸缺乏诱导的子宫内膜蜕膜化异常的体内和体外模型，采用一系列分子生物学技术，围绕细胞自噬的各个环节分析和探讨其分子机制。研究结果将有助于深入阐释叶酸缺乏致胎儿出生缺陷的发生机理，为出生缺陷的预防提供新的思路和线索。

现有证据表明不良妊娠结局与饮食因素密切相关。叶酸由于参与细胞甲基化和DNA合成，在神经管形成和胎儿生长中发挥重要作用，母体叶酸缺乏是胎儿出生缺陷的高危因素。课题组前期研究表明，叶酸缺乏影响子宫内膜蜕膜化进程，但其机制尚不清楚。自噬通过“自我清除”途径维持细胞内环境稳态，研究发现自噬在维持子宫内膜正常功能方面也具有重要作用。因此，本项目将进一步探讨叶酸缺乏如何影响子宫内膜蜕膜化异常以及妊娠维持，并重点关注自噬的变化。. 课题组成功构建了叶酸缺乏孕鼠的子宫内膜体内和体外模型，并按照研究计划完成了全部研究内容，获得以下研究结果：①叶酸缺乏抑制小鼠子宫内膜蜕膜化进程；②叶酸缺乏下调蜕膜细胞自噬水平，且自噬体形成、自噬体与溶酶体融合、内容物降解的各个阶段均受到叶酸缺乏的明显抑制；③自噬诱导剂雷帕霉素可逆转叶酸缺乏对子宫内膜蜕膜化的不良影响，但自噬抑制剂 3-Methy ladenine 无效。并且叶酸缺乏可破坏自噬标志分子与蜕膜化标志分子间的蛋白质相互作用，提示自噬参与调控叶酸缺乏引起的子宫内膜蜕膜化异常；④叶酸缺乏所导致的自噬异常受到 AMPK / mTOR 及 AMPK-SKP2-CARM1 的调控；⑤叶酸缺乏可抑制蜕膜细胞凋亡水平，而雷帕霉素能改善被抑制的细胞凋亡。以上结果表明：孕早期母体叶酸缺乏可通过AMPK / mTOR引发蜕膜细胞自噬紊乱，减少细胞凋亡，干扰子宫内膜蜕膜化进程，导致不良妊娠结局。. 在项目实施过程中，课题组还额外构建了叶酸缺乏的孕鼠胎盘模型、人胎盘外植体模型及HTR8/SVneo 细胞模型进行深入研究，发现：①叶酸缺乏导致孕鼠胎盘内分泌功能紊乱，并抑制胎盘血管生成和细胞分化，最终导致胎盘发育异常；②叶酸缺乏引起孕鼠胎盘自噬紊乱；③自噬抑制剂 3-Methyladenine 可以改善叶酸缺乏引起的胎盘发育异常。以上体内外实验结果证实了自噬参与调控叶酸缺乏引起的胎盘发育异常。 . 总之，本项目首次从细胞自噬角度深入探讨和解析了孕早期母体叶酸缺乏影响子宫内膜蜕膜化进程和胎盘发育功能维持的作用机制，既丰富了对自噬功能的认识，也有助于深入理解叶酸缺乏导致胎儿出生缺陷发生的分子机理，为出生缺陷的预防及治疗提供了新的思路和线索。