摘要
目的:探讨通过优化细胞培养体系,实现Leydig细胞的体外增殖培养。方法:联合应用胶原酶消化、不锈钢滤网过滤及差速贴壁法获得3周龄雄性Wistar大鼠睾丸Leydig细胞,贴壁细胞以DMEM/F12培养液及优化培养体系培养,MTT法、细胞计数法检测培养细胞的增殖能力;分别对原代培养2h、4d细胞进行3β-HSD免疫化学染色及流式细胞术分析,检测细胞成分,同时检测培养细胞睾酮在hCG刺激下分泌能力变化。结果:优化培养体系能够明显促进3周龄大鼠睾丸Leydig细胞大量增殖,群体倍增时间为(2.26±0.31)d,传统培养体系培养细胞群体倍增时间为(16.32±2.14)d,两者差异有显著性(P<0.05);原代细胞经流式细胞术鉴定,3β-HSD阳性细胞所占比例分别为(54.3±7.1)%,培养4d后,增殖细胞3β-HSD阳性细胞率为(93.6±4.6)%。增殖细胞均有睾酮生成功能,在hCG刺激下睾酮分泌均明显上升(P<0.05)。结论:优化培养体系能够促进差速贴壁法获得的睾丸Leydig细胞大量增殖。
Objective:To investigate the feasibility of in vitro proliferation of rat Leydig cells by modifying the cell culture system. Methods: Leydig cells were isolated from three-week-old rats by a procedure combining collagenase dispersion, stainless steel mesh infiltration and differential adhesion. The isolated cells were cultured in DMEM/F12 and modified media for stem cell proliferation, and the proliferation of the cultured cells was evaluated by cell counting and MTT test. The expression of 3β-HSD in the cultured cells was detected by immunohistochemistry and flow cytometry, and testosterone productivity in the isolated Leydig cells with or without hCG stimulation was determined at 2 hours and 4 days after cell isolation. Results: The Leydig cells cultured in the modified media proliferated actively, with a doubling time of (2.26±0.31) days, as compared with (16.32±2.14) days for those cultured in the traditional media (P0.05). The 3β-HSD positive rate in the cultured cells was (554.3±7.1)% after 2 hours and (93.6±4.6)% after 4 days of culture. All the proliferated cells exhibited testosterone productivity, and their testosterone secretion was significantly up-regulated by hCG stimulation (P0.05). Conclusion: Leydig cells isolated by differential adhesion proliferate actively in the modified culture media.
出处
《中华男科学杂志》
CAS
CSCD
北大核心
2011年第2期104-109,共6页
National Journal of Andrology
基金
国家973计划课题(2005CB52270524)
国家自然科学基金(30600619)~~