OAZ1调控鹅卵泡颗粒细胞增殖和凋亡的作用机制研究

卵泡颗粒细胞增殖和凋亡对卵泡发育、闭锁起关键调控作用，其机制尚未被阐明。前期研究发现产蛋期鹅卵巢中鸟氨酸脱羧酶抗酶1（OAZ1）高表达；小黄卵泡颗粒层OAZ1表达量比F1级卵泡高5.56倍。OAZ1通过抑制细胞多胺合成、摄入以及促进Smad1、cyclin D1、Aurora-A和?Np73降解来调控细胞增殖和凋亡。推测OAZ1可调控卵泡颗粒细胞的增殖和凋亡，其机制仍不清楚。因此，本项目利用定点突变技术缺失OAZ1移码位点碱基，制备鹅OAZ1单抗，构建OAZ1基因重组腺病毒过表达和干扰表达载体，转染卵泡颗粒细胞，应用流式细胞术、Real-time PCR、Western blot及高效液相色谱等技术研究高表达、低表达OAZ1对颗粒细胞增殖和凋亡、多胺代谢、FSHR和LHR表达的影响，阐明OAZ1调控鹅卵泡颗粒细胞增殖和凋亡的作用机制，为卵泡发育分子调控机制的研究提供理论依据和新的切入点。

The proliferation and apoptosis of the follicular granulosa cells play key roles in follicular development and atresia, however, the molecular mechanism remains to be investigated. Our previous study found that the level of ornithine decarboxylase antizyme 1 (OAZ1) mRNA expression was higher in the ovaries of the laying geese than in those of the prelaying geese. The level of expression of OAZ1 mRNA in the granulosa layer of small yellow follicles was 5.56 fold higher than in that of F1 follicles.OAZ1 regulates cell proliferation and apoptosis through affecting cellular polyamine metabolism and targeting to degradation Smad1、cyclin D1、Aurora-A and ?Np73. We speculate that OAZ1 should regulate the follicular development by affecting cell proliferation and apoptosis, however, the action mechanism of OAZ1 remains largely unclarified. Therefore, the base at the site of ribosomal frameshift should be deleted by site base mutation method in the project. The monoclonal antibody of OAZ1 should be prepared. The over/interference adenovirus vectors with OAZ1 gene should be constructed, and then transfected into the follicular granulose cells of the geese in vitro.The effect of high and low expression of OAZ1 on cell proliferation and apoptosis, polyamine metabolism, the expression of FSHR and LHR in the granulosa cells should be examined by flow cytometry, real-time PCR, wesern blot and high performance liquid chromatogram, and so on. The mechanism of action of OAZ1 regulating the granulosa cell proliferation and apoptosis should be clarified in geese. The results should provide the theoretical basis and a novel approach for the researches in molecular regulation mechanism of follicular development.