自噬与TGF-β信号通路及低氧间的调控在腭部发育中的功能研究

The most frequent birth defects of craniofacial abnormalities are the congenital cleft lip/palate(CLP). In China, occurrence of CLP estimates 1.82%, which results in complications affecting speech, feeding, hearing, appearance and psychological development. Patients and their families will undergo multiple rounds of surgical repair starting in the first year of life and may continue until 18 or 20 years old. CLP is etiologically heterogeneous and this has critical implications for understanding the biology of facial development, how environmental risks interact with genetic factors and how we can incorporate known etiologic variables to improve clinical care. Autophagy is an evolutionarily conserved cellular process through which long-lived proteins and damaged organelles are recycled to maintain energy homeostasis and is more widely viewed as a basic cell survival mechanism to combat environmental stressors. Disturbance of autophagy may induce craniofacial malformations. Using histological and ultrastructural methods, we investigated the developmental stages of the palate in miniature pigs which was from E26-50 and found autophagosome or autophagosome like structures with variable autophagy associated genes expressions at different stages in palate development. Also we used a previously generating mutant animal model, in which tamoxifen inducible loss of Tgfbr1 in epithelium cells which is more accurately to lead to cleft palate and is differed from previous cleft palate in Tgfbr1 conditional knockout mice model and detected that autophagy associated genes expressions were different from the control in palate. Based on these, we will investigate the mechanism of genetic and environmental triggers on autophagy during mice palate development in vivo and in vitro, especially the crosstalk between autophagy and TGF-β signaling/hypoxia. The study of these genes and their molecular pathway will provide a useful and informative route with which to gain a better understanding of human CLP, and benefits of precise diagnosis, accurate risk assessment and genetic counseling can be achieved. And the potential for treatments and preventative therapies may also become a reality to reduce CLP.

先天性唇腭裂（CLP）是颌面部最常见的发育畸形，在我国发生率为1.82‰，造成患儿颌面畸形，严重影响患儿身心健康，对患者家庭和社会造成很大负担。其病因复杂，遗传因素和环境因素均有重要作用。自噬是机体对体内不利微环境的一种适应机制，其异常可导致发育异常。我们前期研究发现小型猪腭部发育中有自噬体出现，mRNA表达谱分析发现腭部发育各阶段均有自噬相关因子的差异表达；利用我们自己建立的成熟的Tgfbr1诱导性条件敲除小鼠腭裂模型研究发现敲除Tgfbr1后腭裂组织自噬相关蛋白表达异常，本课题将以此为基础，分别从遗传和环境因素着手，在组织和细胞学层面研究自噬对腭部发育的影响，及其与TGF-β信号通路以及低氧在小鼠腭部发育中的调控作用，并寻找其它相关基因，多角度的探讨自噬在腭部发育中的功能机制，寻找关键靶点，从而扩大对唇腭裂发病机制的了解，做好早期预防及诊断治疗，进而减少唇腭裂的发生。

先天性唇腭裂（CLP）是颌面部最常见的发育畸形，在我国发生率为1.82‰，造成患儿颌面畸形，严重影响患儿身心健康，对患者家庭和社会造成很大负担。其病因复杂，遗传因素和环境因素均有重要作用。自噬是机体对体内不利微环境的一种适应机制，其异常可导致颌面发育异常。我们前期研究发现小型猪腭部发育中有自噬体或类自噬体出现，mRNA表达谱分析发现腭部发育各阶段均有自噬相关因子表达；利用我们自己建立的成熟的Tgfbr1诱导性条件敲除小鼠腭裂模型研究发现敲除Tgfbr1后腭裂组织自噬相关蛋白表达异常，本课题以此为基础，分别从遗传和环境因素着手，在组织和细胞学层面研究自噬对腭部发育的影响，及其与TGFβ信号通路以及低氧在小鼠腭部发育中的调控作用，研究发现：（1）Tgfbr1 cKO腭裂小鼠腭部组织与正常对照组小鼠腭部存在自噬与凋亡差异；（2）通过观察自噬促进剂雷帕霉素和抑制剂氯喹对腭突细胞生物学特性的影响，发现自噬可通过影响腭突细胞增殖、凋亡、迁移、成骨影响腭部发育；（3）TGF-β信号通路可通过TGF-β2促进腭突细胞自噬，有可能通过MAPKs通路和核糖体蛋白RPS6Ks蛋白磷酸化影响腭突细胞自噬，从而影响腭部发育；（4）低氧、自噬、糖酵解三者间的平衡调控腭部发育；（5）其他环境因素如尼古丁也可通过影响腭突细胞ROS/ERK通路进而影响自噬从而影响腭部发育，酒精则可通过影响Shh信号通路影响腭部发育；（6）肠道微生物可通过细菌代谢产物经孕鼠血浆、羊水改变胎鼠腭部糖代谢从而影响腭发育；（7）发现维甲酸、Shh信号通路等对颌面发育与再生具有重要作用；（8）前期发现小型猪这种大型动物模型的腭发育较小鼠与人类腭发育过程更为接近，对其腭发育各阶段mRNA和miRNA进行测序分析，获取其表达图谱，筛选出一些潜在靶点，以更好研究人腭部发育基因调控与腭裂发生机制。通过这些研究，增加了自噬对颌面发育作用的了解，发现了糖代谢在颌面部发育中的重要作用，扩大了对细胞分化机制的了解，进一步阐明了先天性唇腭裂的发病机制，为做好早期预防、筛查及诊断，也为将来的基因或药物治疗提供线索，从而减少唇腭裂的发生。

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