OPN5对鸭颗粒细胞凋亡、增殖及类固醇激素生成的影响

旨在研究视神经蛋白（neuropsin，OPN5）对鸭颗粒细胞凋亡、增殖及类固醇激素生成的影响。本研究分别对鸭颗粒细胞进行OPN5过表达质粒和OPN5 siRNA处理72 h （n=6），应用EdU细胞增殖检测技术、流式细胞技术、Annexin V-FITC、RT-PCR、Western blot及ELISA技术系统地检测颗粒细胞的增殖、凋亡情况及生殖相关基因的mRNA、蛋白水平及激素水平的变化规律。结果显示，OPN5过表达能促进鸭卵泡颗粒细胞增殖，抑制鸭卵泡颗粒细胞凋亡；能极显著地促进GnRHR、FSHR、LHR的表达 ，并抑制GnIH、GnIHR的表达（P<0.01）；能显著或极显著上调StAR、CYP11A1、3β-HSD、CYP17A1、CYP19A1的mRNA水平（P<0.05或P<0.01）；显著升高OPN5、3β-HSD及CYP19A1的蛋白表达水平（P<0.05）和极显著升高E2、P4分泌水平（P<0.01），并极显著降低INHβ的水平（P<0.01）。OPN5 siRNA能够显著降低OPN5的表达水平（P<0.01），抑制卵泡颗粒细胞增殖并促进凋亡，极显著下调GnRH、GnRHR、FSHR、LHR（P<0.01），促进GnIH、GnIHR的表达（P<0.01）；并极显著抑制StAR、CYP11A1、CYP17A1的表达（P<0.01）；显著降低OPN5、3β-HSD及CYP19A1蛋白表达水平（P<0.05）及极显著降低E2、P4的分泌水平（P<0.01），并能极显著升高INHβ的水平（P<0.01）。研究表明，OPN5能促进鸭卵泡颗粒细胞的增殖，抑制其凋亡，促进颗粒细胞中类固醇激素的生成和分泌。

Effects of OPN5 on Apoptosis,Proliferation and Steroid Hormone Synthesis of Duck Granulosa Cells

This study aimed to characterize the effects of neuropsin (OPN5) on apoptosis, prolife-ration and steroid hormone production of granulosa cells of duck. The duck granulosa cells were isolated and transfected with OPN5 overexpression plasmid and OPN5 siRNA, respectively. After continuous culture for 72 h (n=6), both proliferation and apoptosis of granulosa cell were detected using EdU cell proliferation assay, flow cytometry, Annexin V-FITC. The mRNA and protein expression levels, as well as hormone secretion were investigated by RT-PCR, Western blot and ELISA, respectively. The results showed that overexpression of OPN5 promoted prolife-ration and inhibited apoptosis of follicular granulosa cells in ducks. Moreover, overexpression of OPN5 significantly promoted the expression of GnRHR, FSHR, LHR, inhibited the expression of GnIH and GnIHR (P<0.01), significantly or highly significantly upregulated the mRNA levels of StAR, CYP11A1, 3β-HSD, CYP17A1 and CYP19A1 (P<0.05 or P<0.01), significantly increased the protein expression levels of OPN5, 3β-HSD and CYP19A1 (P<0.05), highly significantly increased the secretion levels of E2 and P4 (P<0.01). However, the levels of INHβ was highly significantly decreased (P<0.01). OPN5 knockdown with siRNA could effectively downregulate OPN5 expression in transfected cells (P<0.01), inhibit proliferation and promote apoptosis of follicular granulosa cells. siRNA transfection significantly downregulated the expression levels of GnRH, GnRHR, FSHR, LHR (P<0.01), promoted the expression of GnIH and GnIHR (P<0.01), significantly inhibited the expression of StAR, CYP11A1, CYP17A1 (P<0.01). OPN5 knockdown significantly decreased the protein expression levels of OPN5, 3β-HSD and CYP19A1 (P<0.05), highly significantly decreased the secretion levels of E2 and P4 (P<0.01), and highly significantly increased the level of INHβ (P<0.01). In conclusion, OPN5 promotes the proliferation and inhibits the apoptosis of duck follicular granulosa cells, and promotes the production and secretion of steroid hormones in granulosa cells.