新生昆明小鼠睾丸支持细胞和精原细胞的共培养及支持细胞的作用  

Coculture of supporting cell and spermatogonia in testes of neonatal kunming mice and the effect of Sertoli cell

作　　者：张晓丽[1,2] 高英茂[2] 赵舒武[3] 邴鲁军[2] 

机构地区：[1]山东大学附属省立医院生殖中心,济南250021 [2]山东大学医学院组织学胚胎学教研室,济南250012 [3]天津中医药大学组织学胚胎学教研室,天津300193

出　　处：《解剖学报》2009年第2期240-245,共6页Acta Anatomica Sinica

基　　金：教育部高等学校博士学科点专项科研基金资助项目(20010422005)

摘　　要：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 and purify Sertoli cells in testes of 7-8 days postnatal mice and to investigate the influence of Sertoli cells on spermatogonia cultured in vitro by the Co-culture of spermatogonia and Sertoli cell. Methods／The techniques of combined enzymatic digestion,discontinuous percoll gradients centrifugation were used prepare testicular cells suspension. Sertoli cells identified by Sudan IV staining,treated by mitomycin wereserved as feeder cells to coculture spermatogonia with the Sertoli cells layer. Compared to the spermatogonia directly cultured,the size of spermatogonia clones and spermatogonia survival time were observed and recorded under inverted phase contrast microscope,the survival quantity of spermatogonia was determined by MTT assay,and the apoptosis of the spermatogenic cells was detected by Annexin Ⅴ-FITC/PI staining,observed under laser confocal scanning microscope. Difference of the size of spermatogonia clones,survival time,proliferation rate and apoptosis rate were compared between both groups. Results Sertoli cells purity can reach to 95% and above after being identified by Sudan Ⅳ staining. Compared with the control group,the size of spermatoginia clones is larger,spermatogonia survival time is larger and survival quantity is higher,the apoptosis rate is lower. Conclusion Sertoli cells can support the spermatogonia survival and proliferation during the Co-culture in vitro.

关 键 词：支持细胞 精原细胞 饲养层 共培养 小鼠 

分 类 号：R321.1[医药卫生—人体解剖和组织胚胎学]