仔猪睾丸支持细胞的体外生物学特性研究-中国细胞生物学学报.PDFVIP

网络出版时间：2015-08-21 10:42:56 网络出版地址：/kcms/detail/31.2035.q1042.001.html 中国细胞生物学学报 Chinese Journal of Cell Biology 2015, 37(8):1122-1128 DOI: 10.11844/cj cb.2015.08.0145 技术与方法 技术与方法仔猪睾丸支持细胞的体外生物学特性研究 * ( 四川农业大学动物医学院, 温江611130) 摘要 为研究仔猪睾丸支持细胞生物学特性, 以3~5周龄仔猪睾丸为材料, 优化消化和差 异贴壁方法, 分离纯化支持细胞, 研究其体外培养特性包括增殖活力、标志分子和细胞因子的表 达。结果显示, 先以0.25 %胶原酶IV和0.1%透明质酸酶消化60 min, 再以0.25%胰蛋白酶和0.1% DNase I消化20 min, 有利于获得支持细胞; 支持细胞接种后贴壁时间主要集中在3~5 h; 纯化后细 胞胞体宽大, 呈梭形或不规则形; GATA4染色呈阳性, 碱性磷酸酶染色呈阴性; RT-PCR检测, 细胞 表达GATA4、SOX9和INHB , 不表达CYP17、3β-HSD和α-SMA; ELISA检测, 细胞表达GDNF 、LIF 、 CSF-1、EGF 、FGF2, 符合支持细胞的基本特征。 关键词 猪; 支持细胞; 分离; 体外培养; 细胞因子 Characterization of Sertoli Cells from Prepubertal Porcine Testis In Vitro Liu Huanhuan, Yu Shumin*, Xia Juan, Sun Chengliang, Shen Liuhong, Cao Suizhong, Zuo Zhicai, Ma Xiaoping (College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang 611130, China) Abstract To analysis the characterization of sertoli cells in vitro, the modified digestion and differential plating methods were used for isolating sertoli cells from 3~5 week-old porcine testis. Then, cell proliferation, the expression of markers and secretion of cytokines were detected. The results showed that treated with 0.25% IV collagenase and 0.1% hyaluronidase for 60 min, and then with 0.25% trypsin and 0.1% DNase I for 20 min was the optimal method for isolating sertoli cells. And after inoculation, the adhesion process of sertoli cells were accomplished within 3~5 hours. The purified cells were showed with large body and fusiform or irregular shape, and