知识与财富的链接
【目的】采用改良方法对犊牛睾丸支持细胞进行体外快速分离培养,以期建立一种简单快捷的支持细胞原代培养方法。【方法】采用1.0 g/L的Ⅳ型胶原酶和2.5 g/L的胰蛋白酶,对经分离曲细精管和组织剪碎2种方法处理的睾丸组织进行次第消化,比较2种方法对睾丸支持细胞分离效果的影响;采用福尔根染色法和免疫细胞化学方法鉴定睾丸支持细胞。【结果】睾丸组织采用分离曲细精管后进行酶消化的时间(8 min)较组织剪碎法(15min)短,并且前者消化所得有效细胞数(1.0×107)高于后者(0.81×107),两者具有显著差异(P0.05);福尔根染色和免疫细胞化学染色结果显示,分离细胞有卫星核小体存在,且细胞中ABP阳性表达,表明分离培养的细胞确为睾丸支持细胞。【结论】睾丸组织经曲细精管分离后进行胶原酶和胰蛋白酶次第消化,有助于快速高效分离睾丸支持细胞。
【Objective】 We used modified method to separate and culture Sertoli cells in order to set up a simple and effective method for separating Sertoli cells.【Method】 The tissue was treated with the methods of separating seminiferous tubule and cutting tissue respectively.Then,the disuse was digested with the 1.0 g/L collagenase and 2.5 g/L trypsin sequentially.The effect of the two methods on the Sertoli cell separation was compared.Sertoli cells were identified according to Feulgen staining and immunocytochemistry.【Result】 After the seminiferous tubule was separated,the digestion period (8 min) was shorter than that (15 min) after the tissue was treated with cutting,the number of effective cells (1.0×107) was more than that (0.81×107) after the tissue was treated with cutting.The satellite karyosomes were stained in violet.The expression of ABP was examined by SABC staining.【Conclusion】 Separating the seminiferous tubule is an efficient way for the Sertoli cell digested with collagenase and trypsin sequentially.