TSA促进梅花鹿和牛种间克隆胚胎发育的研究
发布时间：2018-10-25 20:25
【摘要】：自从1997年第一只克隆羊多莉出生以后,至今已有19种哺乳动物通过体细胞核移植获得克隆后代,在世界掀起了一阵“克隆”热潮。虽然同属鹿科的赤鹿已经成功被克隆了后代,但是梅花鹿被成功克隆还未报道。梅花鹿是国家一级保护动物,除了可以为人类提供多种名贵的医疗保健药材外,它还具有很高的观赏性和科学研究价值。但是有关梅花鹿克隆的研究处于停滞不前的境地,主要是由于梅花鹿本身数量的减少和屠杀数量的减少,这样就不能提供丰富的卵巢来源,所以极大地限制了梅花鹿克隆胚胎方面的研究进展。所以寻找代替梅花鹿卵母细胞的替代物是梅花鹿种间克隆研究中一个值得关注的关键性问题。本试验探讨了梅花鹿-牛种间克隆胚胎体外培养的条件,试验数据显示梅花鹿胎儿成纤维细胞作为核供体细胞所构建的种间重构胚胎的卵裂率(71.7±4.0%)及囊胚率(13.1±1.7%)显著高于梅花鹿耳成纤维细胞作为核供体细胞的卵裂率(53.8±0.1%)和囊胚率(7.1±0.2%);以280V、脉冲时间10μs、2次直流脉冲条件下进行重构胚胎的细胞融合所获得的梅花鹿-牛种间克隆胚胎的卵裂率(60.9±0.5%)显著高于230V-300V融合电压的其他试验组;牛胚胎培养液SOFAA和梅花鹿胚胎培养液SDSOF对梅花鹿-牛种间重构胚胎的卵裂率没有显著差异,分别是60.7±1.9%及61.7±1.7%,但SDSOF培养基中所获得的种间重构胚胎囊胚率(10.7%±1.2%)显著高于SOFAA培养基中获得的囊胚率(5.6±1.2%)。同时探讨TSA对梅花鹿和牛种间克隆胚胎发育作用。为了更加直观的观察种间胚胎的发育情况,首先建立了梅花鹿pEGFP-N1成纤维细胞系作为核供体细胞。以不处理组为对照组,选择不同浓度的TSA处理核供体细胞24h和种间重构克隆胚胎10h为试验组。结果表明:最适合梅花鹿-牛种间克隆重构胚胎发育的TSA浓度是50nM,筛选的pEGFP-N1成纤维细胞系2及3作为核供体细胞获得的囊胚率显著高于克隆1(9.2%和10.7%vs.4.3%,P0.05)。综上所述,牛卵母细胞可以支持梅花鹿体细胞的重编程及去分化。
[Abstract]:Since the birth of Dolly, the first cloned sheep in 1997, 19 mammals have obtained cloned offspring through somatic cell nuclear transfer, which has caused a wave of "cloning" in the world. Although red deer belonging to the same family have been successfully cloned, the successful cloning of sika deer has not been reported. Sika deer is one of the first class protected animals in the country. In addition to providing a variety of valuable medical and health care materials for human beings, sika deer also has high ornamental and scientific research value. But the research on sika deer cloning is at a standstill, mainly due to the decline in the number of sika deer themselves and the reduction in the number of people slaughtered, which does not provide a rich source of ovaries. Therefore, the research progress in cloned embryos of sika deer is greatly restricted. Therefore, the search for alternative oocytes is a key issue in interspecific cloning of sika deer. In this experiment, the conditions for the culture of sika deer and bovine interspecific cloned embryos in vitro were studied. Experimental data showed that the cleavage rate (71.7 卤4.0%) and blastocyst rate (13.1 卤1.7%) of interspecific reconstructed embryos constructed by sika deer fetal fibroblasts as nuclear donor cells were significantly higher than those of sika deer ear fibroblasts. The cleavage rate and blastocyst rate of nuclear donor cells were 53.8 卤0.1% and 7.1 卤0.2%, respectively. The cleavage rate (60.9 卤0.5%) of sika deer interspecific cloned embryos obtained by cell fusion of reconstructed embryos at 280V and 10 渭 s of pulse time was significantly higher than that of other experimental groups with 230V-300V fusion voltage. There was no significant difference between bovine embryo culture medium (SOFAA) and sika deer embryo culture medium (SDSOF) in cleavage rate of sika deer and bovine interspecific reconstructed embryos, which were 60.7 卤1.9% and 61.7 卤1.7%, respectively. But the rate of blastocysts obtained in SDSOF medium (10.7% 卤1.2%) was significantly higher than that in SOFAA medium (5.6% 卤1.2%). At the same time, the effects of TSA on the development of interspecific cloned embryos between sika deer and cattle were studied. In order to observe the development of interspecific embryos more intuitively, a sika deer pEGFP-N1 fibroblast cell line was established as a nuclear donor cell. The untreated group was used as the control group and the nuclear donor cells were treated with different concentrations of TSA for 24 h and the interspecific reconstructed cloned embryos for 10 h were selected as the experimental group. The results showed that the optimal concentration of TSA for embryo development of sika deer and bovine interspecific clone reconstruction was 50 nm. The blastocyst rate of pEGFP-N1 fibroblasts 2 and 3 as nuclear donor cells was significantly higher than that of clone 1 (9.2% vs 10.7vs.4.3p0.05). In conclusion, bovine oocytes can support the reprogramming and dedifferentiation of sika deer somatic cells.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S814
本文编号：2294746
[Abstract]:Since the birth of Dolly, the first cloned sheep in 1997, 19 mammals have obtained cloned offspring through somatic cell nuclear transfer, which has caused a wave of "cloning" in the world. Although red deer belonging to the same family have been successfully cloned, the successful cloning of sika deer has not been reported. Sika deer is one of the first class protected animals in the country. In addition to providing a variety of valuable medical and health care materials for human beings, sika deer also has high ornamental and scientific research value. But the research on sika deer cloning is at a standstill, mainly due to the decline in the number of sika deer themselves and the reduction in the number of people slaughtered, which does not provide a rich source of ovaries. Therefore, the research progress in cloned embryos of sika deer is greatly restricted. Therefore, the search for alternative oocytes is a key issue in interspecific cloning of sika deer. In this experiment, the conditions for the culture of sika deer and bovine interspecific cloned embryos in vitro were studied. Experimental data showed that the cleavage rate (71.7 卤4.0%) and blastocyst rate (13.1 卤1.7%) of interspecific reconstructed embryos constructed by sika deer fetal fibroblasts as nuclear donor cells were significantly higher than those of sika deer ear fibroblasts. The cleavage rate and blastocyst rate of nuclear donor cells were 53.8 卤0.1% and 7.1 卤0.2%, respectively. The cleavage rate (60.9 卤0.5%) of sika deer interspecific cloned embryos obtained by cell fusion of reconstructed embryos at 280V and 10 渭 s of pulse time was significantly higher than that of other experimental groups with 230V-300V fusion voltage. There was no significant difference between bovine embryo culture medium (SOFAA) and sika deer embryo culture medium (SDSOF) in cleavage rate of sika deer and bovine interspecific reconstructed embryos, which were 60.7 卤1.9% and 61.7 卤1.7%, respectively. But the rate of blastocysts obtained in SDSOF medium (10.7% 卤1.2%) was significantly higher than that in SOFAA medium (5.6% 卤1.2%). At the same time, the effects of TSA on the development of interspecific cloned embryos between sika deer and cattle were studied. In order to observe the development of interspecific embryos more intuitively, a sika deer pEGFP-N1 fibroblast cell line was established as a nuclear donor cell. The untreated group was used as the control group and the nuclear donor cells were treated with different concentrations of TSA for 24 h and the interspecific reconstructed cloned embryos for 10 h were selected as the experimental group. The results showed that the optimal concentration of TSA for embryo development of sika deer and bovine interspecific clone reconstruction was 50 nm. The blastocyst rate of pEGFP-N1 fibroblasts 2 and 3 as nuclear donor cells was significantly higher than that of clone 1 (9.2% vs 10.7vs.4.3p0.05). In conclusion, bovine oocytes can support the reprogramming and dedifferentiation of sika deer somatic cells.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S814
【参考文献】
中国期刊全文数据库 前2条
1 ;Serial nuclear transfer improves the development of interspecies reconstructed giant panda (Aluropoda melanoleucd) embryos[J];Chinese Science Bulletin;2002年06期
2 殷宗俊;龙学仁;许瑞锋;徐福乐;朱建儿;王霖;;受体牛胎次与黄体等级对胚胎移植妊娠率的影响[J];中国奶牛;2006年07期
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