新生牛生精上皮细胞体外培养的研究

两步酶消化法制备新生牛生精上皮细胞悬液,分离纯化、鉴定精原干细胞和支持细胞,冷冻保存支持细胞。以支持细胞为饲养层探索不同培养条件下精原干细胞的增殖情况,免疫组化鉴定。结果表明:10%DMSO与0.1mol/L的海藻糖组合作为冷冻保护剂,冷冻效果较好;血清浓度为2.5%、支持细胞密度为5×105个/mL时,新生牛精原干细胞较易形成集落;精原干细胞及其集落的AKP染色和C-kit免疫组化均呈阳性。

Study on in vitro Culture Spermatogenic Epithelial Cells of New Born Calf

The spermatogenic epithelial cell suspension was obtained through two-step enzymatic digestive method from new born calf,spermatogonia stem cells and sertoli cells were isolated,purified and identified,and sertoli cells were cryopreserved with different cryoprotectants.Feeder layer was made of sertoli cells,the proliferation of the spermatogonia stem cells in different culture conditions was studied,spermatogonia stem cells was identified by immunohistochemistry.The results showed that basic medium adding 10%DMSO and 0.1 mol/L trehalose would increase the resuscitative rate of sertoli cells significantly,the culture condition with 2.5% serum and 5 ×105/mL sertoli cells made spermatogonia stem cells form colony easily;Identification of C-kit and AKP were positive by immunohistochemistry.