原代睾丸支持细胞分离纯化及双室培养模型的建立  

Establishment of Dual-Chamber Model for Rat Sertoli Cells Primary Culture

作　　者：周元陵[1] 钟先玖[1] 吴鑫[1] 钱海雷[2] 金泰廙[2] 

机构地区：[1]复旦大学附属金山医院,上海200540 [2]复旦大学公共卫生学院,上海200032

出　　处：《环境与职业医学》2005年第3期208-211,共4页Journal of Environmental and Occupational Medicine

基　　金：上海市金山区卫生局科研基金(编号:98-区卫-3)

摘　　要：[目的]通过建立双室培养模型,为探讨毒物或药物对睾丸支持细胞作用机制提供一个合适的体外研究方法。[方法]18d龄雄性SD大鼠,脱颈椎处死后,无菌条件下取睾丸组织,经胰蛋白酶和胶原酶二步消化后获得支持细胞,经接种、纯化和鉴定,获得纯度>95%支持细胞。培养液A为DMEM培养基和F12培养基按照1:1比例用去离水配制,每升培养基中含有20万单位青霉素和0.2g链霉素,用2%的碳酸氢钠调整pH值为7.2左右。培养液B为培养液A中加入维生素A、E(200ng/ml)和胰岛素(2μg/ml)。培养液C为B培养液中再加入卵泡刺激素(100ng/ml)和睾丸酮(10-8mol/L)。将制备好的支持(Sertoli)细胞混悬液按2.5×106个/ml浓度接种于24孔培养板内,培养条件为35℃(睾丸细胞的适宜温度),2%CO2恒温静止培养。测定3种不同培养液中培养的支持细胞形成的跨细胞上皮电阻值(transep-ithelialelectricalresistance,TER)。[结果]培养液加入维生素A、E和胰岛素后,支持细胞形成的TER有所升高,而培养液在加入睾丸酮和卵泡刺激素后,Sertoli细胞形成的TER则显著升高。培养第1天3种培养液中培养的Sertoli细胞形成的TER分别为33.85、31.38和34.26Ω/cm2,培养第13天分别为198.53、289.91和707.50Ω/cm2。[结论]Sertoli细胞双室培养模型可模拟体内状态的“血睾屏障”,为体外研究雄性生殖毒性机制提供一个合适模型。[Objective] To explore the mechanisms in effects of toxicants and drugs on male reproductive system and set up Ser-toli cells primary dual-chamber culture model. [Methods] Testis tissue was obtained from 18-day male rats which were killed by dislocation of cervical vertebra. Sertoli cells, purity exceeding 95% after treatment, were obtained from the two-step digestion with enzyme of testis tissue, and cultured in three different culture media. Medium A consists of DMEM and F12(1:1) confecting with deionized water. Per liter of medium A included 200,000 units of penicillin and 0.2 g streptomycin, and the value of pH of medium A was adjusted by sodium hydrogen carbonate with concentration of 2% . Medium B was prepared by adding vitamin A (200 ng/ml), vitamin E (200 ng/ml) and insulin (2 μg/ml) to medium A. Medium C was prepared by adding follicle stimulating hormone (100 ng/ml) and testosterone (10-8mol/L) to medium B.The prepared suspension of Sertoli cells was inoculated in cultivating plate of 24 wells with concentration of 2.5×106 cell/ml. The condition of cell culture was 35℃ in the air with 2% CO2. The value of transepithelial electrical resistance (TER) in the dual-chamber system was determined in different media. [Results] TER value of Sertoli cells was increased when vit A, vit E and insulin were added to culture media, and TER value of Sertoli cells was significantly increased when testosterone and follicle stimulating hormone were added to culture media. In the first day, TER valur of Sertoli cells in different media was 33. 85,31. 38 and 34.26 Ω/cm2,respectively. 13 days later.TER value of Sertoli cells in different media was 198.53,289.91 and 707.50 Ω/cm2,respectively . [Conclusion] The dual-chamber culture system of Sertoli cells could mimic the blood testis barriers in vivo, and it could be used as a suitable model in reproductive toxicology.

关 键 词：睾丸 支持细胞 原代培养 双室模型 

分 类 号：R114[医药卫生—卫生毒理学] R135[医药卫生—公共卫生与预防医学]