山羊iPS细胞诱导及培养体系的优化  

Optimization of Induced and Cultivation System of Goat iPS Cells

作　　者：邱峰龙[1] 左其生[1] 李东[1] 李伟[1] 陈庭锋[1] 韦光辉[1] 李碧春[1] 

机构地区：[1]扬州大学动物科学与技术学院,扬州225009

出　　处：《畜牧兽医学报》2014年第5期740-749,共10页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：转基因生物新品种培育重大专项(2011ZX08008-003);国家高技术研究发展计划(2011AA100307-04);江苏高校优势学科建设工程资助项目

摘　　要：为了高效、持续的诱导获得并培养山羊诱导性多能干细胞(Induced pluripotent stem cells,iPS),本研究从饲养层和培养液方面进行优化。将分离获得3代以内的小鼠胎儿成纤维细胞(Mouse embryonic fibroblast,MEF)、山羊胎儿成纤维细胞(Goat embryonic fibroblast,GEF)用丝裂霉素C处理后,分别按1×105 mL-1 MEF、1×105mL-1 GEF、5×104 mL-1 MEF+5×104 mL-1 GEF的密度接种,以高糖DMEM+20%胎牛血清(FBS)和KnockoutDMEM+20%血清替代物(KSR)为培养液,研究不同饲养层种类和培养液对山羊iPS细胞获得和培养的影响。结果显示,山羊iPS细胞在接种密度为5×104 mL-1 MEF+5×104 mL-1 GEF的饲养层上,使用KnockoutDMEM+20%KSR组合的培养液,山羊iPS细胞的诱导效率以及消化传代后的克隆形成率均极显著高于其他5组(P<0.01)。对该组培养的山羊iPS细胞进行碱性磷酸酶(AKP)染色呈阳性;Oct4、SSEA-1、Tra-1-60、Tra-1-81免疫荧光检测呈阳性;RT-PCR检测有Oct4、Sox2、Klf4和Nanog基因的表达;体外能分化形成类胚体。结果表明,将细胞接种在密度为5×104 mL-1 MEF+5×104 mL-1 GEF的饲养层上,使用KnockoutDMEM+20%KSR的培养液更适合山羊iPS细胞的获得和培养。本试验为山羊iPS细胞的体外研究、临床试验、动物基因组修饰和山羊胚胎干细胞(Embryonic stem cells,ES)的建系奠定基础。In order to induced and cultured the goat iPS cells efficiently and continuous,the feeder cells and culture conditions were optimized in this study.MEF and GEF cells were inoculated at concentrations of 1×105 mL-1 MEF,1×105 mL-1 GEF and 5×104 mL-1 MEF+5×104 mL-1 GEF as the feeder layers of goat iPS cells after they were treated with Mitomycin C within 3passages,and cultured in DMEM+20%FBS,KnockoutDMEM+20%KSR,observed the different influence.The results showed that the induction efficiency and formation rates after digest of goat iPS cells in 5 ×104 mL-1 MEF+5×104 mL-1 GEF and Knockout DMEM+20% KSR were more higher than those cultured on the other 5groups(P<0.01).Growth states of goat iPS cells were observed in this group and undifferentiated phenotypes were detected,included expression of alkaline phosphatase,and dected the expression of Oct4,Sox2,Klf4,Nanogand cell marker Oct4,SSEA-1, Tra-1-60,Tra-1-81by RT-PCR.In the condition of feeder layer free,goat iPS cells differentiation were observed in vitro.The results indicated that the acquisition and cultivate of goat iPS cells in 5×104 mL-1 MEF+5×104 mL-1 GEF and KnockoutDMEM+20% KSR were more easily thanthose cultured on the other 5groups.This study established a foundation for further study on iPS cell in vitro studies,clinical trials,animal genome modification and goat ES cells line.

关 键 词：饲养层 培养液 IPS细胞 山羊 

分 类 号：S827[农业科学—畜牧学]