关键词: 染料木素, 大豆苷元, 牦牛, 卵巢颗粒细胞, 增殖, 凋亡

Abstract:

【Background】Soybean isoflavones, mainly including genistein and daidzein, could exert estrogen-like effects, scavenge free radicals and promote cell proliferation. Granulosa cells are an important cell population in follicles and its physiological state is directly related to ovarian function, and the apoptosis of granulosa cells causes oocyte atresia. Yak (Bos grunniens) is one of the endemic species in Qinghai-Tibet plateau, but its reproductive rate is so low, however, the mechanism is still unclear. Ovary granulosa cell is an ideal model to study the regulating mechanism of female animal reproduction. 【Objective】The aim this study was to investigate the effects of soybean isoflavones on the proliferation and apoptosis of granulosa cells of yak ovary, and to provide evidence for the mechanism of soybean isoflavones.【Method】The yak ovarian granulosa cells were isolated and cultured. Immunohistochemistry staining was used to check FSHR for ovarian granulosa cell authenticating. MTT assay was used to detect cell proliferation, then the growth curve was drawn. Ovarian granulosa cells treated with different concentration of genistein and daidzein (0, 1 000, 2 000, 3 000, 4 000 and 5 000 pg·mL^-1), the optimal concentration of genistein or daidzein for was selected to treat the second-generation granulosa cells for 48 h, then, the cell culture medium was collected and used to detect the concentration of estradiol and progesterone secreted by granulosa cells by ELISA. At the same time, the cells were collected to extract total RNA and to research the expression of proliferation-related gene AKT1 and apoptosis-related genes Bcl-2, Bax, Bad, Fas, FasL, Caspase-3 and p53 by qRT-PCR.【Result】The yak ovarian granulosa cells were typical epithelial-like cells. After inoculated for 2 h, the granulosa cells began to adhere to the wall. After 12 h, the cells appeared aggregation and growth. After 24 h, the cells were larger and showed long fusiform, star-shaped or polygonal. After cultured for 48 h, the cells looked like paving appearance. Immunohistochemistry staining showed FSHR positive indicated that the cultured cells were ovarian granulosa cells. MTT assay showed that the growth curve of the yak ovarian granulosa cells was S-shape: the growth was slow in 24 h and the cells were in the latent growth stage, and then, which was rapidly proliferated at 24-48 h and entered the exponential growth phase. The granulosa cells, in the plateau phase, steadily proliferated during 48-120 h. After 120 h, the density of living cells began to decline, and the cells entered the phase of degeneration. The second-generation of granulosa cells grew faster and the exponential proliferation phase at 24 h, so the second-generation of granulosa cells was selected for this experiment. MTT assay showed that treated with 3 000 pg·mL^-1 genistein or daidzein for 48h, the cell viability were significantly promoted (P<0.01), the secretion of estradiol were induced treated with daidzein, but the progesterone secretion were markedly inhibited treated with genistein. The results of qRT-PCR showed that 3 000 pg·mL^-1 genistein or daidzein significantly up-regulated the expression of AKT1, Bcl-2, Bad, p53, Fas and Bcl-2/Bax (P<0.01), down-regulated the expression of Bad and FasL. In addition, genistein significantly down-regulated the expression of Caspase-3 and daidzein significantly up-regulated it (P<0.01). 【Conclusion】The yak ovarian granulosa cells were isolated and cultured to provide an effective cell model for further study on the yak female reproduction regulating mechanism. The results indicated that genistein and daidzein inhibited the progesterone secretion of yak ovarian granulosa cells, promoted cell proliferation, and protected cells from apoptosis by up-regulating Bcl-2, p53, Fas and Bcl-2/Bax and down-regulating Bad and FasL.

Key words: genistein, daidzein, yak, ovarian granulosa cells, proliferation, apoptosis