抽提物处理供体细胞对克隆胚胎发育的影响  

Effects of Donor Cells Treated with Egg Extracts on the Development Competence of Cloned Embryos

作　　者：郭芹芹 范宗兴[1] 郝海生[1] 刘岩[1] 赵学明[1] 朱化彬[1] 杜卫华[1] 

机构地区：[1]中国农业科学院北京畜牧兽医研究所,北京100193

出　　处：《中国畜牧兽医》2016年第2期477-486,共10页China Animal Husbandry & Veterinary Medicine

基　　金：国家科技支撑项目(2012BAD12B01-2);基本科研业务费重点项目(2013ywf-zd-2);家畜胚胎工程与繁殖创新团队(ASTIP-IAS06-2015)

摘　　要：本研究采用爪蟾卵母细胞抽提物处理和牛胎儿成纤维细胞（JBCFF）,并以发生重编程的细胞为核供体进行体细胞核移植,研究其对克隆效率的影响。分别超排3只爪蟾,收集卵母细胞后提取其抽提物,用BCA蛋白浓度测定试剂盒确定其蛋白含量,SDS-PAGE分析其中所含蛋白的种类。应用细胞膜透化剂Digitonin对建立的JBCFF进行通透处理和PI染色,筛选最适浓度;并用获得的抽提物处理牛体细胞,获得重编程细胞。以发生重编程的体细胞为供体进行核移植,同时比较离子霉素＋6-DMAP和A23187＋6-DMAP两种组合激活后克隆胚的体外发育能力。结果,3个爪蟾卵母细胞抽提物样品的蛋白浓度分别为56.2255、64.6570和71.2158μg/mL,其中所含蛋白种类一致,主要集中在40-55、70-100ku之间。经过连续的筛选,透化剂Digitonin对JBCFF通透处理的最适浓度为7μg/mL,PI染色显示透化效率为55.44%。爪蟾卵母细胞抽提物与体细胞共孵育后继续培养6-7d,细胞聚集形成＂克隆簇＂。分别以＂克隆簇＂细胞和未处理细胞为核供体,制备的重构胚在融合率（92.83%和96.04%）、卵裂率（89.64%和89.78%）和囊胚率（24.06%和23.12%）均无显著差异（P〉0.05）;离子霉素＋6-DMAP和A23187＋6-DMAP两种激活方式对克隆胚胎的分裂率（92.16%和92.28%）和囊胚率（23.21%和24.18%）均无显著影响（P〉0.05）。爪蟾卵母细胞抽提物诱导和牛体细胞发生重编程,并恢复到较低的分化状态,但没有显著促进克隆胚胎的体外发育,可见缺少对胚胎发育起重要作用的重编程过程,还需要继续深入研究。Japanese Balck cattle fetal fibroblasts（JBCFF）were induced with Xenopus leavis egg extracts and somatic cell nuclear transfer（SCNT）was carried out with the reprogrammed JBCFF as donor cells in order to investigate their effects on SCNT efficiency.Three samples of egg extracts were acquired from different Xenopus laevis.The protein contents and kinds in extracts were evaluated with BCA Protein Quantification Kit and SDS-PAGE.Concentration of Digitonin to permeabilize JBCFF was optimized and assessed with PI staining.Reprogrammed cells treated with egg extract were used as donor in SCNT.Additionally the reconstructed embryos were activated with ionomycin＋6-DMAP and A23187＋6-DMAP to compare their effects on the development competence.The protein contents of extracts samples were 56.2255,64.6570 and 71.2158μg/mL,respectively,the each extract had the same composition about 40-55 and 70-100 ku.The optimal concentration of Digitonin was 7μg/mL and the permeabilization rate was 55.44%.After extracts treatment and continuous culture for 6-7d,JBCFF formed well-defined colony structures.No significant composition difference was found in rates of fusion（92.83% vs 96.04%）,cleavage（89.64% vs 89.78%）and blastocyst formation（24.06% vs 23.12%）of cloned embryos when the colony cells and JBCFF without extracts treatment were used as donor cells（P〈0.05）.Similarly,the two activation methods had no significant effect on the developmental competence of cloned embryos（cleavage rate 92.16% vs 92.28%,blastocyst rate 23.21% vs 24.18%）.Conclusively,Xenopus leavis,egg extracts could induce JBCFF reprogramming to a low differentiated state.However donor cells with reprogramming partially could not improve the development of cloned embryos and its mechanism requires further research.

关 键 词：爪蟾 卵母细胞抽提物 供体细胞 重编程 克隆胚胎 

分 类 号：S852.65[农业科学—基础兽医学]