鸡精原干细胞定向诱导分化特性的研究  

Orientate-Derivation Character of Chicken Spermatogonial Stem Cells

作　　者：李碧春[1,2] 魏彩霞[2] 余飞[2] 何先红[2] 倪黎纲[2] 陈强[2] 王宵燕[2] 肖小君[2] 徐琪[2] 刘铁铮[1] 

机构地区：[1]江苏省农业科学院畜牧研究所,南京210014 [2]扬州大学动物科学与技术学院,扬州225009

出　　处：《畜牧兽医学报》2008年第10期1336-1342,共7页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金(30371031);高等学校博士学科点专项科研基金(20061117004);国家高新技术“863”项目(2007AA100504)

摘　　要：取孵化18～20d的鸡胚睾丸，分离精原干细胞（SSCs），培养传代3次后，用特异性化学物质定向诱导分化，以研究鸡培养传代后SSCs保持多向分化的潜能。①用特异的化学物质地塞米松、β-磷酸甘油钠、维生素C诱导鸡SSCs向成骨细胞分化，用Von Kossa＇s法、改良的钙钴法及免疫组化法进行成骨细胞鉴定；②用维甲酸（RA）、3-异丁基-1-甲基黄嘌呤（IBMX）诱导SSCs向神经元样细胞分化，甲苯胺蓝特异染色和组织化学鉴定。③用地塞米松、胰岛素、3-异丁基-1-甲基黄嘌呤（IBMX）诱导SSCs向脂肪细胞分化，特异性的油红O染色和脂肪细胞标志基因PPAR7检测。结果显示，SSCs被诱导15～21d后分化为成骨细胞，诱导形成率为80％；Von Kossa＇s染色细胞间布满黑色颗粒，具有矿化基质沉积；改良钙钴法碱性磷酸酶染色胞浆呈深棕色或深黑色，免疫组化染色细胞呈阳性。SSCs被诱导3～7d后分化为神经元样细胞，甲苯胺蓝染色阳性率为85％。SSCs被诱导7～21d后，分化成脂肪细胞，分化率为85％。诱导的脂肪细胞经特异性的油红O染色为阳性，并高表达脂肪细胞标志基因PPARγ。结论：在不同的诱导条件下，SSCs可被定向诱导分化为3种成体细胞，证明其培养传代后仍具有多向分化能力。The aim of this study was to investigate the capability of spermatogonial stem cells （SSCs） differentiating into osteoblasts, neuron-like cells, and adipocytes in vitro. In present study, SSCs were isolated from testis of chicken embryo on 18^th-20^th hatching days, then cultured and subcultured to the 3rd passage in vitro. （1） SSCs were Orientate-induced into osteoblasts by medium consisting of desamethason, glycerol 2-phosphate disodium salt hydrate and vitamin C, and differentiated cell were examined with Von Kossa＇s stain, cytochemistry staining and immunohistochemistry stain. （2）SSCs were Orientate-induced into neuron-like cells in the medium containing RA and IBMX. The differentiated cells were identified by toluidine blue and immunohistochemistry stain. （3）SSCs were induced into adipocytes in the medium consisting of desamethason, insulin and IBMX. The differentiated cells were then identified by oil red-O and specific express PPARγ. The results showed that 80% SSCs were induced into osteoblasts during 15-21 d after cultured in induction medium. The osteohlasts formed Von Kossa stain positive nodules. Dark granules were found in the cell, suggesting the deposition of mineralized materials. The osteo blasts showed brown or deep dark after alkaline phosphatase stain and showed Collagen I positive. About 85% cells was toluidine blue positive after 7 d when SSCs were induced into neuron like cells. About 85% adipocytes. The induc oil red-O positive cell formed during 14-21 d when SSCs were induced into ed cells were highly expressed PPARγ. Therefore, we concluded that chicken SSCs have the capability of multi differentiation in vitro.

关 键 词：精原干细胞 成骨细胞 神经元样细胞 脂肪细胞 诱导 分化 

分 类 号：S831[农业科学—畜牧学] S831.11[农业科学—畜牧兽医]