小鼠精原干细胞体外培养的研究

目的建立精原干细胞与Sertoli共培养细胞模型,探讨Sertoli支持精原干细胞扩增的可能机制。方法以7—8 d龄小鼠为材料,Percoll密度梯度离心,并将分离后的生精细胞接种至Sertoli饲养层上,37℃下共培养,在倒置相差显微镜下观察细胞的生长特点,并对培养15 d的细胞进行苏木精-伊红染色、C-Kit受体检测。结果①增殖细胞的生长特点及形态学特征与文献报道精原干细胞的特征相符合。②苏木精-伊红染色显示培养的生殖细胞大小均匀,核大而圆着色深。C-Kit受体显示细胞膜呈强阳性,背景基质细胞为阴性。这些均符合精原干细胞的生物学特征。结论精原干细胞在未加入细胞因子的Sertoli饲养层中能较长时间生长并分裂增殖。

Research on culture In vitro of the mouse spermatogonial stem cells

Objective To construct cell mode] of spormatogonial stem cells (SSCs) with Sertoli cells layer,and study the possible mechanism.Methods Germ cells were isolated from testes of KM mice aged 7-8 days,and were en- riched using Percoll grads centrifugation;Sertoli cells treated with mitomycin C were used as feeder cells,the culture medi- um was DMEM supplemented with 5% FBS,co -culture was done at 37℃.Cell growth was observed under inverted phase contrast microscope.Cultured 15 -day cells were treated with haematoxylin-eosin staining (HE) staining and C-kit re- ceptor test.Results①The growth eharacteristics and morphological characteristics of proliferating ceils were accorded with those reported in literatures.②HE staining showed that the size of reproductive cells was even with big round deep- stained nucleus,C-Kit receptor test showed strong positive cell membrane and negative matrix cell,which met the biologi- cal characteristics of SSCs.Conclusion SSCs can grow divide and proliferate in feeder layer without cytokine and primary cultured Sertoli cells.