OAZ1调控鹅卵泡颗粒细胞增殖和凋亡的作用机制研究

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.

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。

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，OAZ1可调控卵泡颗粒细胞增殖和凋亡，其机制仍不清楚。本项目转染突变型OAZ1过表达和干扰表达载体，研究OAZ1调控鹅卵泡颗粒细胞的作用及机制，主要获得以下结果：（1）多胺在鹅卵泡发育过程中具有重要调控作用，高浓度多胺可促进小黄卵泡发育成排卵前等级卵泡，而低浓度的多胺可加速机体对排卵后卵泡的吸收；（2）OAZ1不仅能诱导细胞内多胺反馈调节，维持细胞多胺稳态，而且还可通过Cyclin-D1，Bax、Bcl-2和Caspase 3信号转导途径调控鹅卵泡颗粒细胞的增殖和凋亡；（3）OAZ1基因表达水平发生变化时，主要影响细胞内腐胺和亚精胺浓度；（4）OAZ1可介导卵泡颗粒细胞FSHR、LHR、ER和PRLR基因表达以及E2合成、分泌来参与调控鹅卵泡发育、排卵及闭锁过程。项目实施期间，共发表SCI论文2篇；CSCD核心库收录论文9篇；申请国家发明专利5项，其中1项已获得授权；培养硕士研究生2名。