Tgfbr1 诱导性条件敲除小鼠腭裂发生的机制探讨

The most frequent birth defects of craniofacial abnormalities are the congenital cleft lip/palate(CLP). The majority of CLP (70%) are regarded as nonsyndromic, where the clefts occur without other anomalies. In China, occurrence of CLP estimates 1.82%, which results in complications affecting appearance, feeding, speech, hearing 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. Combined with multigenic inheritance and the influence of non-genetic factors, identifying the key genes in human CLP represents a major challenge. The TGF-β family is particularly interesting in palate development and isoforms 1, 2 and 3 are all expressed during this process. Altered TGF-β signaling causes syndromic and nonsyndromic cleft palate. Inducible conditional knockout mice models are one of the most accurate models to investigate gene function. Previously we generated a mutant animal model, in which tamoxifen inducible loss of Tgfbr1 in epithelium cells which is more accurately leads to cleft palate and is differed from previous cleft palate in Tgfbr1 conditional knockout mice model. Based on this mice model, we will investigate the molecular mechanism of TGF-β signaling-mediated palatogenesis by analyzing the role of TGF-β1,2,3 and the signaling downstream target genes both in SMAD-dependent and SMAD-independent in vivo and in vitro. As the role of miRNAs in craniofacial development is beginning to emerge, we will also investigate their roles in this mice model. And we will also investigate the role of Tgfbr1 during mouse embryonic palatal mesenchymal (MEPM) cells differ into muscle cells or osteogenic cells. 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）是颌面部最常见的先天性发育畸形，非综合征性唇腭裂占其中70%，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。其病因比较复杂，TGF-β信号通路是其中研究最多的通路之一，诱导性条件敲除小鼠是目前研究基因功能最为精确的模型之一。我们前期利用他莫昔芬在上皮诱导性条件敲除Tgfbr1（Tgfbr1 cKO）得到了一个不同于以往Tgfbr1条件敲除小鼠腭裂表型的腭裂模型，本课题将以此为基础，分别在组织学和细胞学层面研究TGF-β经典和非经典通路以及miRNA在Tgfbr1 cKO腭裂发生中的作用，并寻找其它相关基因，同时分析Tgfbr1对腭部间质细胞分化的影响，多角度的探讨Tgfbr1诱导性条件敲除小鼠腭裂发生的作用机制，从而增加对TGF-β通路的认识，扩大对唇腭裂发病机制的了解，进一步做好早期预防及诊断，进而减少唇腭裂的发生。

先天性唇腭裂（CLP）是颌面部最常见的先天性发育畸形，在我国发生率为1.82‰，居出生缺陷前五位，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。动物模型是研究唇腭裂发病机制的主要手段，诱导性条件敲除小鼠是目前研究基因功能最为精确的模型之一。我们前期利用他莫昔芬在上皮诱导性条件敲除Tgfbr1（Tgfbr1 cKO）得到了一个不同于以往Tgfbr1条件敲除小鼠腭裂表型的腭裂模型，本课题以此为基础，对TGF-β信号通路在腭部发育的功能进行了研究，发现：（1）抑制TGFBR1后，腭突细胞增殖下降，凋亡增高，成骨及成软骨分化受到抑制，体内研究也发现敲除Tgfbr1发生腭裂的小鼠腭突细胞凋亡增加，TGF-β经典和非经典通路均参与该过程；（2）通过基因芯片筛查，发现miR-376c-3p可能与腭裂发生有关。miR-376c-3p抑制腭突细胞增殖和成骨分化，荧光素酶实验显示miR-376c-3p可能通过与Tgfbr1结合抑制其表达，从而影响腭部发育；（3）诱导性条件敲除Tgfbr1小鼠出现牙发育滞后以及牙矿化异常，体外实验发现在小鼠牙胚发育钟状期，TGFβ3和TGFBR1（又名ALK5）促进牙胚间充质细胞（TGMCs）增殖和成牙/骨向分化。利用SD208阻断TGFBR1表达，TGMCs增殖削弱, 成牙/骨向分化受到抑制；TGF-β经典通路和非经典通路均参与该过程；（4）获得了较完整的小型猪腭部发育时相和组织学、超微组织特征，发现小型猪胚胎腭部发育在时相上较小鼠更接近于人，可作为研究腭部发育的大型哺乳动物模型；（5）在小型猪和小鼠腭部发育组织中发现了自噬体的存在，通过研究自噬促进剂雷帕霉素和抑制剂氯喹对小鼠腭突细胞生物学特性的影响，发现自噬可以影响腭部发育；（6）环境因素如酒精可通过影响腭突细胞增殖凋亡分化等影响腭部发育；（7）炎症微环境对口腔颌面部发育、干细胞分化都具有重要影响。大麻素受体CB1可通过p38 MAPK 和 JNK信号通路促进炎症状态下牙周膜干细胞PDLSCs成骨/牙分化，该工作为研究炎症状态下颌面部干细胞的分化调控机理提供了线索。通过这些研究，增加了对颌面部发育机制的认识，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。