全反式维甲酸修复Hoxa1-/-猪外耳缺陷的营养学及遗传学机理研究

External ear malformation not only negatively influence the appearance of animals, but also hurt their hearing and health. Elucidation of the mechanism underlying outer ear deformity formation and repairing has been one of the key researches in human medicine. A congenital external ear genetic defect pedigree has been constructed with Erhualian and Shaziling pigs, and the responsible gene-hoxa1 and its causal mutation underlying this defect has been identified with the previous project of “Identification of genetic defect causative gene for pig congenital outer ear development”(31260540) that was financially supported by National Natural Scinece Foundation of China. In this study, the HOXA1-/- pig model will be used to compare the effect of rescuing outer ear malformation with all-trans retinoic acid offered to pregnant sow at different time and level,with the aim to achieve the optimal feeding time and level of all-trans retinoic acid by phenotypic experiment and CT scan. Then, a feeding experiment with all-trans retinoic acid added at the optimal time and level will be conducted to study the influence of all-trans retinoic acid on the expression and signal pathway of the causative hoxa1 gene using RNA-Seq, Real-time PCR and immunofluorescence staining. The results would reveal the mechanism of all-trans retinoic acid rescuing the outer ear deformity of HOXA1-/- pig and provide novel insights into studying and treating congenital genetic outer ear malformations for humans.

外耳畸形不仅影响动物的表型美观，还影响其听力和健康，外耳畸形的形成与修复机制研究是人类医学研究的重点之一。申请人所在的实验室在国家自然科学基金“猪先天性外耳发育遗传缺陷致病基因的鉴别”项目（31260540）的资助下，利用“二花脸×沙子岭”猪先天性外耳遗传缺陷病家系，鉴别到了导致猪出现先天性外耳遗传缺陷的致病基因(HOXA1)及其因果突变位点。本项目以HOXA1-/-基因型猪为模型，采用表型观察和CT扫描，比较全反式维甲酸在不同补饲时间和剂量条件下修复HOXA1-/-基因型猪外耳畸形的效果差异，确定全反式维甲酸的适宜补饲时间和剂量，并以此进行饲养试验，采用RNA-Seq、Real-time PCR和免疫荧光染色等方法研究全反式维甲酸对HOXA1-/-基因表达和信号通路的影响，揭示维甲酸修复HOXA1-/-猪外耳畸形的分子机制，为人类先天性外耳遗传缺陷病的研究与治疗提供重要借鉴。

Hoxa1基因发生g.50111251 G > TC突变会导致猪胎儿耳朵发育出现异常，形成畸形耳，同时畸形耳小猪出生后伴随着呼吸困难和早期死亡等现象，解剖后发现其肺充血肿胀。本项目进行了在不同妊娠时间（配种后12、13和14天）给妊娠母猪补饲不同剂量（0、4、5和6 mg/kg体重）全反式维甲酸的饲养试验，通过外耳评分、CT扫描、ELASA检测、代谢组、微生物组和转录组等方法研究了全反式维甲酸对hoxa1-/-基因型胎猪耳畸形修复和肺发育的影响。结果表明，在母猪配种后的第14天按6 mg/kg体重的剂量一次性给母猪补饲全反式维甲酸能有效减少hoxa1-/-基因型胎猪畸形耳的形成和异常胎肺的发生，提高与肺正常发育相关基因的表达。这一研究成果对防治胎儿宫内发育迟缓、胎儿畸形耳形成和初生胎儿呼吸异常具有积极的参考价值。