大鼠Sertoli细胞与精原细胞的分离及共培养

目的 分离纯化14 d大鼠Sertoli细胞与精原细胞,用Sertoli细胞作为饲养层对大鼠精原细胞进行体外培养研究.方法 酶消化法制备大鼠睾丸组织单细胞悬液,采用牛血清白蛋白(BSA)不连续密度梯度沉降法分离Sertoli细胞和精原细胞.结果 经分离的Sertoli细胞与精原细胞的纯度分别达到92.73%和78.36%.Sertoli细胞与精原细胞共培养,可见精原细胞发生分裂增殖等行为.结论 在添加EGF、bFGF和GDNF等细胞因子的10%胎牛血清DMEM/F12培养基中,精原细胞能够存活一定时间;而Sertoli细胞表现为旺盛的生长状态,并可促进精原细胞的分裂与增殖.

SEPARATION AND CO-CULTURE OF SERTOLI CELLS AND SPERMATOGONIAL CELLS FROM RAT TESTIS

Objective To isolate and purify Sertoli cells and spermatogonia from rat testis,and to study the proliferation and differentiation of the spermatogonia Co-cultured with sertoli cells. Methods After enzymatic digestions of rat testis,the suspension passed the BSA uncontinuous gradient medium in a gravity unit by velocity sedimentation.Then the spermatogonia were further purified by different times of attachment culture.Results The purities of Sertoli cells and spermatogonia were 92.73% and 78.36% respectively after velocity sedimentation separation.The semi-anchored spermatogonial cells were usually round or oval in outline,singly scattered or existed as aligned,clumped populations,while the outline of the attached Sertoli cells in plate was fibroblast-like cells with little protrusions.Conclusion The results suggest that the spermatogonial cells can survive and proliferate for some time in the culture medium containing EGF,bFGF and GDNF,while Sertoli cells can be more prolific and enhance the mitosis and proliferation of the spermatogonial cells.The confluent monolayer of Sertoli cells can be used as feeding layers for Co-culture with spermatogonial cells.