不同培养系统对鸡精原干细胞体外增殖作用的比较  

Comparison of Cell Proliferation in Chicken(Gallus gallus)Spermatogonial Stem Cells In vitro Under Several Culture Conditions

作　　者：施青青[1] 左其生[1] 李东[1] 张蕾[1] 黄晓梅[1] 张振韬[1] 张亚妮[1] 李碧春[1] 

机构地区：[1]扬州大学动物科学与技术学院/江苏省动物繁育与分子设计重点实验室,扬州225009

出　　处：《农业生物技术学报》2014年第6期736-743,共8页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金项目(No.31272429;No.31301959);高等学校博士学科点专项基金新教师类资助课题(No.2012325012000);江苏省普通高校研究生科研创新计划项目(No.CXLX12-0928);江苏高校优势学科建设工程资助项目

摘　　要：本研究比较了层粘连蛋白(laminin)、纤维连接蛋白(fibronectin)、胶原蛋白(collagen)以及睾丸支持细胞(sertoli cell)4种不同培养系统对鸡(Gallus gallus)精原干细胞(spermatogonial stem cells,SSCs)体外增殖的作用效果。采用三酶两步消化法分离SSCs,细胞经明胶差速纯化后培养,将传至3代的SSCs重新接种于层粘连蛋白、纤维连接蛋白和胶原蛋白包被的培养皿中以及睾丸支持细胞制备的饲养层上,通过形态学、5-乙基-2'-脱氧尿嘧啶核苷(EDU)细胞增殖、qRT-PCR技术检测不同培养系统对鸡精原干细胞体外增殖的作用效果。结果表明,鸡SSCs的碱性磷酸酶(AKP)阳性克隆数在睾丸支持细胞组最高,达到每视野(32±3)个,层粘连蛋白组、纤维连接蛋白组和胶原蛋白组分别为(26±3)、(24±2)和(23±2)个,三组差异不显著(P>0.05);EDU检测睾丸支持细胞组细胞增殖率为(92.82±2.15)%,显著高于三组基质蛋白组(P<0.01);qRT-PCR结果显示,增殖标记基因原癌基因(myconcogene,c-myc)、Kruppel样因子4(kruppel-like factor 4,Klf4)、增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)在睾丸支持细胞组的表达量最高,其次为层粘连蛋白组,c-myc和Klf4在纤维连接蛋白组中的表达要高于胶原蛋白组,PCNA则相反。实验结果表明:三组基质蛋白及睾丸支持细胞都能够促进鸡SSCs的体外增殖,其中睾丸支持细胞作用最佳,其次为层粘连蛋白,纤维连接蛋白和胶原蛋白两者作用效果差异不显著。该结果为进一步优化鸡胚SSCs的体外培养体系,阐明生殖细胞增殖机制提供了实验基础和理论支撑。This study aimed to compare the in vitro effects of laminin, fibronectin, collagen, and coculture with sertoli cells on the efficiency of chicken （Callus gallus） spermatogonial stem cells （SSCs） proliferation.Chicken SSCs were isolated by two-step enzyme digestion and purified by gelatin differential adherent. Three passaged SSCs were inoculated on laminin, fibronectin and collagen coated culture dishes and co-culturedwith sertoli cells. The efficiency of cell proliferation under each culture condition was detected by alkaline phosphatase activity（AKP）, EDU（5-ethynyl-2＇-deoxyuridine） staining and quantitative polymerase chainreaction （qPCR）. Results showed that the number of SSCs AKP positive clones was highest in sertoli cellsgroup with （32±3） clones each vision, while that of laminin group, fibronectin group and collagen group were （26±3）, （24±2） and （23 ±2）, respectively, and the later three groups showed no significant difference （P〉0.05）;EDU staining showed cell proliferation rate in sertoli cell group reached （92.82± 2.15）%, which was significantly higher than that of other groups （P〈0.01）; qRT-PCR results showed that the expression ofproliferation marker genes such as c-myc（myconcogene）, K/f4（kruppel-like factor 4） and PCNA（proliferating cell nuclear antigen） was highest in sertoli cells, followed by laminin group, the expression of c-myc and Klf4in fibronectin group were higher than that of collagen group, while PCNA was on the contrary. The results indicated that both matrix proteins and sertoli cells could promote the proliferation of chicken SSCs in vitro,and sertoli cells was identified as the most appropriate factor for in vitro cell proliferation, followed by laminin, fibronectin and collagen showed no significant difference （P〉0.05）. The results provide theoreticaland experimental references for the further optimization of chicken SSCs culture system and understanding of SSCs proliferation mechanism in vitro.

关 键 词：胶原蛋白 纤维连接蛋白 层粘连蛋白 睾丸支持细胞 鸡 精原干细胞 

分 类 号：S963.214[农业科学—水产养殖]