兔卵巢颗粒细胞分离培养条件的优化

Optimization of Isolation and Culture Conditions for Rabbit Ovarian Granulosa Cells

本研究通过比较不同培养条件下兔卵巢颗粒细胞的生长状态、增殖情况,了解兔卵巢颗粒细胞体外生长增殖的特性,优化兔卵巢颗粒细胞体外培养体系。选用2只4~6月龄新西兰白兔母兔肌肉注射孕马血清促性腺激素并于6 h后处死,采集卵巢颗粒细胞,在体外培养至贴壁后利用卵泡刺激素受体免疫荧光染色鉴定兔颗粒细胞。进一步分别在含5%、10%、15%血清的DMEM培养基和DMEM/F-12培养基中培养颗粒细胞,观察细胞生长状况,并使用CCK-8法测定细胞在不同浓度血清及培养基条件下的增殖状况。结果显示:随着血清浓度的提高,测得的OD值不断增加并在15%浓度下趋于平稳,表明在一定程度上增加血清浓度有助于颗粒细胞增殖;兔卵巢颗粒细胞在DMEM/F-12培养基中的增殖效果优于DMEM培养基。因此,兔卵巢颗粒细胞在含有15%血清的DMEM/F-12培养基中增殖状况最好。

In order to understand the growth characteristics of rabbit ovarian granulosa cells in vitro and optimize the culture system of rabbit ovarian granulosa cells,the growth status and proliferation of rabbit ovarian granulosa cells under different culture conditions were compared.New Zealand female rabbits aged 4-6 months were selected and injected with PMSG intramuscularly and sacrificed 6 hours later.Granulosa cells from the ovary were collected and cultured in vitro when adhesion of the rabbit ovarian granulosa cells were observed,immunofluorescence staining was used to identify.Furthermore,granular cells were cultured in DMEM medium and DMEM/F12 medium containing at serum concentration of 5%,10%,and 15%,respectively.In order to observe cell growth,and CCK-8 method was used to determine cell proliferation under different conditions.The results showed that with the increase of serum concentration,the measured OD value is also increasing and tends to be stable at 15%concentration,indicating that increasing the serum concentration to a certain extent is helpful for the proliferation of granulosa cells;The proliferation effect of granulosa cells in DMEM/F-12 medium is better than that in DMEM medium.Therefore,granulosa cells proliferate best in DMEM/F-12 medium containing 15%serum.