水牛卵巢颗粒细胞的分离培养及凋亡测定

Isolation,Culture and Apoptosis Determination of Buffalo Ovarian Granulosa Cells

研究主要是通过比较水牛卵巢颗粒细胞在不同培养基中的生长状态,并对细胞的增殖、核型及凋亡情况进行检测,以了解水牛卵巢颗粒细胞体外生长特性,建立水牛卵巢颗粒细胞的体外培养体系。结果发现,分离获得的水牛卵巢颗粒细胞存活率约为60%;采用DMEM培养基,颗粒细胞的生长速度和生长状态优于TCM-199和DMEM/F12培养基;细胞培养24h后开始零星增殖,3～5d增殖速度达到高峰;第1、3、5、7代颗粒细胞正常核型比率差异不显著,均在85%以上;第1代颗粒细胞的凋亡率与第5代差异显著(P<0.05),与第7代差异极显著(P<0.01)。结果表明,DMEM培养基更适宜用于水牛颗粒细胞的体外培养;水牛颗粒细胞能稳定地进行传代培养,染色体的数目不会发生明显改变,但细胞凋亡率会随着培养代数的增加而明显升高。

The purpose of this study is to compare the growth of buffalo ovarian granulosa cells （GCs） cultured with differ ent culture medium, and research the proliferation, karyotype and apoptosis of GCs to know well the growth characteristic of GCs in vitro, so that to establish a culture system of buffalo ovarian GCs in vitro. The results showed; the viability of GCs was about 60% after isolation. The growth speed and density of GCs were more dominant as culturing in DMEM medium than that of TCM-199 and DMEM/F12 medium. The GCs began to proliferate after cultured for 24 h, and reached a proliferation peak during cultured for 3 to 5 d. Karyotype analysis displayed that beyond 85% of GCs had normal karyotype in the 1, 3, 5, 7 passage, which did not show that significant difference among these passages. The apoptosis ratio of the 5 and 7 passages of GCs was higher as comparing with the 1 passage （P〈0.05, P%0.01）. These results indicated that the DMEM medium was more suitable for GCs culturing in vitro, and the GCs could be passaged and keep relatively stable karyotype, but the apoptosis ratio would be elevate with the increase of the passage in vitro culture.