GnIH和INH基因疫苗联合免疫大鼠和牛的效果及作用机制探讨

Our previously studies have proved that there are some difference in the immune responses of little and big animal immunized inhibin DNA vaccine. Recently, gonadotropin-releasing inhibiting hormone (GnIH) could inhibit FSH secretion of pituitary, and Inhibin (INH) has a similar physiological role. But the mechanism of their interaction is unclear. This project would construct vaccines of GnIH gene fusion expression and GnIH-INH gene co-expression by using vaccine vector system which waas used to constructing INH gene vaccine in our laboratory. Then, three vaccines were alone and combined immuned for comparing the follicular development, oogenesis, ovulation, fecundation, litter size, reproductive, endocrine changes and the effects of newborns in rats and cattle. The objects of this project are designed to: (1) clarify the effects and the difference of the follicular development, oogenesis in rat and cattle immunized by INH and/or GnIH vaccines; (2) clarify the relationship and regulatory mechanism between INH, GnIH, gonadotropin-releasing hormone, melatonin, kiss peptide, follicle-stimulating hormone, luteinizing, estradiol and progesterone in rat and cattle; (3) further improve the INH gene immunization and promote application of the technology. Successful implementation of this project would provide theoretical guiding significance for the rational use of reproductive endocrine hormones, and study the new technology for improving the fertility.

我们前期研究发现，卵泡抑制素（INH）基因疫苗的免疫效果在大、小动物之间存在差异，推测与生理代偿作用有关。促性腺激素释放抑制激素（GnIH）是近期发现抑制垂体FSH分泌的激素，与INH有类似的生理作用，但其相互作用与机制尚不清楚。本项目应用前期构建的INH基因疫苗质粒系统，构建GnIH基因融合表达疫苗和GnIH-INH基因共表达疫苗。然后，将这3种疫苗单独和联合免疫大鼠和牛，比较各种免疫对鼠和牛卵泡发育、卵子发生、排卵、受胎、产仔、生殖内分泌变化及后代生长发育的效果，旨在：①阐明INH和GnIH对牛和鼠卵泡发育和卵子发生的作用及其在单、双胎动物之间的差异；②阐明INH、GnIH、促性腺激素释放激素、褪黑激素、吻肽、促卵泡素、促黄体素、雌二醇和孕酮等激素的相互作用规律；③进一步完善INH基因免疫技术，促进该技术的推广应用。本项目实施成功后，有助于合理利用生殖内分泌激素，开发促繁殖新技术。

本项目系统的研究了GnIH对雄鼠生殖及生殖内分泌的影响及其可能的分子调控机制，发现GnIH能抑制小鼠睾丸精子发生和类固醇激素的生成。首次在小鼠卵巢颗粒细胞中发现GPR 147的表达，RFRP-3能够在体外直接作用于小鼠卵巢颗粒细胞，通过降低颗粒细胞中FHSR mRNA和P-ERK1/2蛋白的表达来降低颗粒细胞孕酮的合成及存活率，从而间接影响卵泡发育。成功构建了p-SINH、p-SINH/SRFRP和p-TPA-SINH/ TPA-SRFRP三种基因疫苗，免疫小鼠后产生了明显的免疫反应，提高了产仔数；免疫绵羊后能够产生明显的免疫反应，提高绵羊外周血中促性腺激素水平。将抑制素基因疫苗通过肌肉注射和鼻饲免疫免疫水牛均能刺激机体产生抗抑制素抗体，促进水牛发情和排卵，提高受胎率，免疫效果呈剂量依赖关系。肌肉注射与鼻饲免疫抑制素基因疫苗均有较高的抗体阳性率，且在非繁殖季节，以肌肉注射组的发情、排卵和受胎率较高，与繁殖季节相当。抑制素基因免疫对水牛体温、心率和牛场周边环境没有不良影响。本项目的顺利实施有助于进一步了解GNIN和INH的功能，揭示GNIH调控动物生殖内分泌的作用机制，促进激素的合理利用，进一步开发提高动物繁殖力的基因免疫技术，促进其推广应用。