供体细胞处理方法对水牛核移植效果的影响  

Effect of donor cell treatment on development of somatic cell nuclear transfer embryos in buffalo

作　　者：陆凤花[1] 石德顺[1] 韦英明[1] 成俊萍[1] 韦精卫[1] 潘红平[1] 

机构地区：[1]广西大学动物繁殖研究所,广西南宁530005

出　　处：《广西农业生物科学》2006年第B09期193-194,共2页Journal of Guangxi Agricultural and Biological Science

基　　金：国家863计划项目(2002AA206651);广西科学基金(桂科青0640004)

摘　　要：本研究探讨了水牛供体细胞不同培养处理方法对细胞周期及其核移植效果的影响。流式细胞仪分析供体细胞周期的结果显示,水牛胎儿耳皮成纤维细胞或颗粒细胞,在增长期、汇合长满期或经血清饥饿培养或0.1μg/mL蚜栖菌素(APD)+0.5%胎牛血清(FBS)培养处理后,G0+G1期细胞比率差异显著(P<0.05),其中以0.1μg/mL APD+0.5%FBS培养处理的G0+G1期细胞比率最高(87.89%和89.04%);而增长期的G 2+M期的细胞比率以及汇合长满期的S期细胞比率明显高于其他三组,但经血清饥饿处理后,DNA碎片明显高于其他三组(P<0.05)。当分别以0.1μg/mL APD+0.5%FBS培养处理后的水牛胎儿耳皮成纤维细胞或颗粒细胞作供核时,重组胚的卵裂率及囊胚发育率,均显著高于血清饥饿处理或汇合长满期的同种供体细胞(成纤维细胞:70.14%vs 58.29%和55.90%,21.33%vs 10.55%和8.72%;颗粒细胞:74.35%vs 60.51%和57.61%,22.17%vs10.26%和9.78%,P<0.05),但汇合长满期或血清饥饿处理的供体细胞构建的重组胚,其卵裂率和囊胚率均没有显著差异(P>0.05)。将经APD处理后的供体细胞构建核移植胚胎,移植给受体水牛后有1头受体水牛妊娠并成功的产下后代。以上结果表明:0.1μg/mL APD+0.5%FBS预培养处理供体细胞,能有效的抑制细胞停留在G0/G1期,并能提高其核移植胚胎的发育率,而且已成功的获得后代。但在本培养系统中,血清饥饿处理供核细胞是没有必要的。The success of nuclear transfer is thought to be principally dependent on transfer of nuclei in G0 or G1 phase. In this study, the cell cycle characteristic of buffalo fetal fibroblasts and granulosa cells cultured in cycling, culture to confluency, serum-starvation and a reversible cell cycle inhibitor aphidicolin Aphidicolin （APD） 0.1 μg/mL＋0.5% fetal bovine serum （FBS） treatment were investigated. This was obtained by using flow cytometry to measure cellular DNA content and calculated the percentages of cells in G0＋G1, S, and G2＋M phase. Moreover, the developmental ability of buffalo embryos derived from fetal fibroblasts and granulosa cells that cmtureu to confluency, serum-starvation and 0.1 μg/mL APD ＋0.5% FBS treatment were examined. As a result, both buffalo fetal fibroblasts and granulosa cells, cycling culture resulted in lower percentages of cells in G0＋G1 （52.12% and 50.07%）, but significantly higher percentages of cells in G0＋G1when cells treated with 0.1 μg/mL APD＋0.5% FBS （87.89% and 89.04%） than cultured to confluency （66.03% and 63.18%） and serum-starvation （77.87% and 76.57%） （P〈0.05）. Conversely, cycling cells contained more percentages of cells in G2＋M than other treatments, and cultured to confluency resulted in higher percentages cells existing in S phase. However, serum starvation induced more percentages of DNA fragmentation than other treatments （P〈0.05）. The rate of cleavage and blastocysts development of embryos reconstructed with fetal fibroblasts or granulosa cells treated with 0.1 μg/mL APD＋0.5 %FBS were significantly higher than that of serum starvation group and culture to confluency group （fibroblasts： 70.14% vs 58.29%o and 55.90%, 21.33% vs 10.55% and 8.72% ; granulose： 74.35% vs 60.51% and 57.61%, 9.2.17% vs 10.26% and 9.78%, p〈0.05）, however there was not significantly different in the cleavage rate and embryonic development of blastocysts between embryos derived from serum starvation and culture to co

关 键 词：水牛 体细胞核移植 细胞周期 胚胎 

分 类 号：S823.83[农业科学—畜牧学] Q813[农业科学—畜牧兽医]