鸡贮精腺上皮细胞的分离及原代培养

试验旨在探讨稳定可靠的贮精腺上皮细胞分离及原代培养方法,为研究鸡贮精机理提供细胞模型。以鸡输卵管的子宫阴道交接部组织样为材料,采用酶消化法和组织块培养法分离培养母鸡贮精腺上皮细胞,观察母鸡贮精腺上皮细胞的培养情况,比较不同细胞培养方法获得贮精腺上皮细胞的生长情况。结果表明,用胶原酶或胰酶单独消化母鸡子宫阴道交接部组织,经100目过滤后获得的贮精腺上皮细胞24 h后可贴壁,但48～72 h后细胞死亡;用胶原酶Ⅺ(0.01 g/mL)与胰酶(0.25%)先后消化母鸡子宫阴道交接部组织后再经100目过滤获得的贮精腺上皮细胞贴壁性良好,24～48 h细胞出现明显增殖,72 h后细胞增殖速度减慢,开始死亡;用组织块培养法7 d可获得鸡贮精腺上皮原代细胞,该细胞可传2～3代;用组织块培养法获得的细胞进行免疫组化试验,发现细胞表达贮精腺差异表达基因编码的NXPH1蛋白,该蛋白在培养细胞内的表达符合其分泌蛋白特性,表明组织块培养法所获细胞可用于后续研究。综上,用组织块培养法获得的鸡贮精腺上皮细胞可为研究母鸡贮精腺机制提供细胞模型。

Isolation and Primary Culture of Epithelial Cells of Sperm Storage Tubules in Hens

This study was aimed to discuss the stable and reliable method of isolation and primary culture of epithelial cells of sperm storage tubules (SSTs),and provide a cell model for studying the mechanism of chicken sperm storage.The uterovaginal junction of the chicken oviduct was used to culture the epithelial cells of sperm storage tubules with enzyme digestion and explant culture methods.The culture of SSTs cells in hens and the growth of the cells isolated with different cell culture methods was observed.The results showed that after the uterovaginal junction of hen was digested with collagenase or trypsin and then filtered with 100 mesh, the epithelial cells of SSTs could adhere to wall after 24 h,but the cells died after 48-72 h.After the uterovaginal junction of hen was digested with collagenase Ⅺ (0.01 g/mL) and trypsin (0.25%) and then filtered with 100 mesh,there was obvious proliferation at 24 to 48 h,the cells slowly proliferated after 72 h,and then began to die.The epithelial cells of SSTs could be obtained with tissue block culture for 7 days,and the cells could be transferred for 2 to 3 generations.Immunohistochemistry was used to test whether the protein of differentially expressed neurexophilin 1 (NXPH1) of chicken SSTs was located in the primary cells with tissue explant culture.The results showed that NXPH1 expressed in the nuclei and cytoplasm of the cultured cells,indicating that the primary cells with tissue explant culture could be used in subsequent experiments.In conclusion,the epithelial cells of sperm storage tubules obtained with explant culture could provide a cell model for studying the mechanism of sperm storage in hens.