小鼠精原干细胞的分离培养及相关标记物检测

发布时间：2019-01-22 10:15

【摘要】：目的建立一套简单高效的小鼠精原干细胞(SSCs)分离及体外培养体系,检测相关干细胞标记物的表达,为将SSCs用于抗肿瘤干细胞药物的筛选及鉴定奠定基础。方法采用组织化学法比较DBA/2乳鼠(6～8天龄)和成年鼠(26～28周龄)曲细精管解剖结构的特点。选取乳鼠睾丸制备原代细胞,比较不同消化酶的消化效率。利用差异贴壁法即睾丸体细胞在明胶上贴壁而精原细胞不贴壁的特点,富集SSCs。比较以StemPro34和α-MEM为基础培养基的培养体系及明胶、血清等因素对细胞贴壁及生长性能的影响,观察不同培养时间的细胞形态,免疫荧光法鉴定SSCs及肿瘤干细胞(CSCs)相关标记物的表达。结果乳鼠睾丸中精原细胞含量相对较高,胰蛋白酶消化后能获得较多数量的原代细胞。在以StemPro34为基础培养基,含1%胎牛血清及明胶铺板的条件下体细胞贴壁较充分。分离的精原细胞在以丝裂霉素C处理过的体细胞为饲养层的环境中生长良好,呈现出典型的SSCs生长特性,13 d左右可形成明显的细胞球。免疫荧光结果显示,SSCs标记物胶质细胞源性神经营养因子(GDNF)家族受体α1(GFRα1)及VASA蛋白在细胞球中高表达;CSCs标记物CD44在As、Apr、Aal各期细胞及细胞球的内部细胞中均有表达,而体细胞中则几乎不表达。结论初步建立了DBA/2小鼠SSCs的分离及体外培养体系,CD44在发育早期的精原细胞中表达较强。
[Abstract]:Objective to establish a simple and efficient (SSCs) isolation and culture system for mouse spermatogonial stem cells and to detect the expression of related stem cell markers in order to provide a basis for the screening and identification of antitumor stem cell drugs using SSCs. Methods the anatomical characteristics of the seminiferous duct of DBA/2 neonatal rats (68 days old) and adult rats (26 weeks old 28 weeks old) were compared by histochemical method. Primary cells were prepared from the testis of newborn mice and the digestibility of different digestive enzymes was compared. Using differential adherent method, that is, testicular somatic cells adhered to gelatin but spermatogonia did not adhere to the wall, enriched SSCs.. The effects of culture system, gelatin and serum on cell adhesion and growth performance on StemPro34 and 伪-MEM culture medium were compared, and the cell morphology of different culture time was observed. The expression of (CSCs) related markers of SSCs and tumor stem cells was identified by immunofluorescence assay. Results the content of spermatogonium in testis of neonatal rats was relatively high, and a large number of primary cells could be obtained after trypsin digestion. Under the condition of 1% fetal bovine serum and gelatin board, the adherent of somatic cells was more adequate on StemPro34 medium. The isolated spermatogonia grew well in the feeder layer of somatic cells treated with mitomycin C and showed typical SSCs growth characteristics. The results of immunofluorescence showed that the glial cell-derived neurotrophic factor (GDNF) family receptor 伪 1 (GFR 伪 1) and VASA protein were highly expressed in the cell spheres. CD44, a CSCs marker, was expressed in the cells and the inner cells of the spheres of As,Apr,Aal at all stages, but almost not in the somatic cells. Conclusion the isolation and in vitro culture system of SSCs from DBA/2 mice were established. CD44 was expressed strongly in spermatogonial cells at the early stage of development.
【作者单位】：中国医学科学院北京协和医学院医药生物技术研究所肿瘤室;
【基金】：中央级公益性科研院所基本科研业务费专项(IMBF201006)~~
【分类号】：R329

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