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1、106中国兽医学报2009年1月第29卷第1期ChinJVetSciJan.2009Vol.29No.1细胞松弛素B对水牛体细胞核移植效果的影响3陆凤花,覃琦丽,黄华,罗婵,成俊萍,石德顺,韦英明(广西大学动物繁殖研究所,广西南宁530005)摘要:以水牛耳皮成纤维细胞为供体细胞,采用电融合方法,探讨细胞松弛素B(CB)对水牛体细胞核移植效果的影响。体外成熟培养22～24h的水牛卵母细胞去核后,将经0.1mg/LAphidicolin(APD)+0.5%FBS培养2～9d的水牛耳皮成纤维细胞注射到卵周隙中再经电融合(100V/mm,15μs,电脉冲
2、3次)构建核移植重构胚。重构胚经化学激活后(5μmol/L)离子霉素5min,2mmol/L62DMAP3h)培养,7～9d评定其胚胎发育能力。结果显示,在含CB(3mg/L)的融合液中进行电融合后,核移植的融合率、重组胚的存活率、卵裂率和囊胚率与对照组(不含CB)相比均无显著差异(P>0.05);核移植重组胚激活前用含CB(6mg/L)的培养液培养1h,其激活后的存活率(97.52%)和体外囊胚发育率(22.09%)均显著地高于未经CB处理的重组胚的存活率(93.87%)和囊胚率(13.25%,P<0.05);重组胚经离子霉素激活5min后,在6
3、2DMAP+CB中培养3h的分裂率明显低于放在62DMAP中培养3h的分裂率(65.37%vs78.92%,P<0.05),但囊胚发育率无显著差异(11.19%vs10.96%,P>0.05)。这表明水牛体细胞核移植电融合时,融合液中不添加CB,而核移植重组胚激活前经CB培养处理后,有利于胚胎的进一步发育,但激活后用CB培养处理会降低胚胎的发育率。关键词:水牛;体细胞;核移植;电融合;细胞松弛素B中图分类号:S823183文献标识码:A文章编号:100524545(2009)0120106204EffectofthecytochalasinBont
4、hedevelopmentofbuffalosomaticcellnucleartransferembryos3LUFeng2hua,QINQi2li,HUANGHua,LUOChan,CHENGJun2ping,SHIDe2shun,WEIYing2ming(AnimalReproductionInstitute,GuangxiUniversity,Nanning530005,China)Abstract:EffectofthecytochalasinB(CB)onthedevelopmentofbuffalosomaticcellnuclear
5、transfer(NT)em2bryoswereinvestigatedinthisstudy.Buffalooocytesobtainedfromovariesatslaughterwerematuredinvitrofor22224handthenenucleatedinmanipulationmediumcontaining5mg/LcytochalasinB.FibroblastsinDMEM+10%FBSweretreatedwith0.1mg/Laphidicolin(APD)for1dayand0.5%FBSfor229dayswer
6、etransferredintoenu2cleatedoocytesbyelectronicfusion(100V/mm,15μsand3pulses).Thereconstructedembryoswereactivatedbyexposuringthemto5μmol/Lionomycinfor5minandthenculturingin2mM62DMAPfor3h.Afteractivation,em2bryoswereculturedin30μLdropofgranulosacellmonolayersfor729days,toevalua
7、tetheembryonicdevelopment.AdditionofCB3mg/Ltoelectrofusionmanipulationmedium,therewasnosignificantdifferenceinthefusionrate,survivalrate,cleavagerateandblastocystdevelopmentofthenucleartransferembryosascomparedwiththecontrolgroup(P>0.05).Treatmentofreconstructedembryoswith6mg/
8、LCBfor1hbeforeactivationresultedinmoreembryossurvivedanddevel
