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目的分离、纯化、培养小鼠睾丸支持细胞,通过与精原细胞共培养,研究体外培养的支持细胞对精原细胞的影响。方法用复合酶消化、密度梯度离心和差异贴壁法分离、纯化、培养新生小鼠睾丸支持细胞,以苏丹Ⅳ染色鉴定支持细胞;用丝裂霉素-C处理支持细胞后作为饲养层,与精原细胞共培养;以直接分离培养的精原细胞作对照。倒置相差显微镜观察,细胞培养第3天,四甲基偶氮唑盐(MTT)法测定精原细胞的增殖率;膜联蛋白Ⅴ-异硫氰酸荧光素/磷脂酰肌醇(ANNEXIN-Ⅴ-FITC/PI)染色;激光扫描共焦显微镜观察记录精原细胞的凋亡率,比较精原细胞克隆大小、存活时间、增殖率和凋亡率的不同。结果分离、纯化后经苏丹Ⅳ染色鉴定的小鼠支持细胞纯度可达95%以上,与支持细胞共培养的精原细胞的克隆大小、存活时间和细胞增殖率明显高于对照组,而凋亡率则明显低于对照组。结论支持细胞可以促进精原细胞的增殖,抑制其凋亡。
OBJECTIVE: To isolate, purify and culture mouse testis-supporting cells and study the effect of cultured cells on spermatogonia by co-culturing with spermatogonia. Methods Sertoli cells of newborn mice were isolated and purified by compound enzyme digestion, density gradient centrifugation and differential adherent method. The supporting cells were identified by Sudan Ⅳ staining. After treated with mitomycin C, Spermatogonial cells were co-cultured; spermatogonia were directly isolated and cultured as control. Inverted phase contrast microscope was used to observe the proliferative rate of spermatogonia by MTT assay on day 3 of cell culture. Annexin V-fluorescein isothiocyanate / phosphatidylinositol (ANNEXIN-Ⅴ -FITC / PI) staining. The apoptosis rate of spermatogonia was observed by laser scanning confocal microscopy. The spermatogonia clone size, survival time, proliferation rate and apoptosis rate were compared. Results After isolation and purification, the purity of the mouse support cells identified by Sudan Ⅳ staining was over 95%. The size, survival time and cell proliferation rate of spermatogonia co-cultured with the support cells were significantly higher than those of the control group, but apoptosis The rate was significantly lower than the control group. Conclusion Support cells can promote the proliferation of spermatogonia and inhibit its apoptosis.