【作者】 李彦欣；

【导师】 李宁；

【作者基本信息】 中国农业大学 ， 生物化学与分子生物学， 2004， 博士

【摘要】 在保护濒危动物和进行核质互作分析方面，异种克隆技术是非常有效的方法。本研究利用牛卵母细胞作为受体，通过显微操作与牦牛或羚牛体细胞构建异种重构胚，将牦牛的异种重构胚移植到代孕牛中，获得了妊娠，并有望于2004年6—7月间出生。此外本研究还对异种克隆技术和异种体细胞核重编程进行了初步探讨。 1 供体细胞的培养：本实验从北京动物园、青海互助县、秦皇岛野生动物园分别采集了羚牛和牦牛组织样，建立了21个细胞系。细胞系种类包括耳成纤维细胞、输卵管上皮、颗粒细胞系三种。实验证明样品采用0℃保存48小时，对细胞系的建立无负面影响。这为野外收集珍稀动物组织，建立细胞系提供了切实可行的方法。 2 体细胞克隆技术基础平台的建立：首先，在去核方法上，本实验比较了H33342染色法，盲吸法和高渗法并结合这三种方法作了优化，形成本实验的去核方法：其次，本实验利用牛卵母细胞孤雌激活的方法，比较了四种激活方法，发现利用复合激活方法比单个化学试剂激活效率高(83.6、68.7：45、62.5)，而在复合激活的方法中以A23187+6-DMAP激活效率较高(分裂率，83.6)；最后，通过比较M199培养液和CR1aa培养液对孤雌激活胚胎的培养效率，发现CR1aa培养液较适合牛胚胎的培养。 3 异种克隆牦牛囊胚研究：本实验利用不同个体的牦牛耳成纤维细胞、颗粒细胞和输卵管细胞，分别作为供核细胞移入去核牛卵母细胞中，均可完成体外牦牛的早期囊胚发育(囊胚率高达35％)。通过对不同年龄阶段、不同个体、不同细胞类型、细胞的饥饿与否、细胞冷冻与否，以及卵母细胞成熟率，不同融合到激活时间对克隆效率的比较发现：卵母细胞的质量是克隆成功的基础，当成熟率在40％以下时，将显著影响异种克隆牦牛的囊胚发育(由25％降到3％，P＜0.01)；影响异种克隆牦牛早期囊胚发育的重要因素为延迟激活时间，当延迟激活1.5小时可显著提高异种克隆囊胚率(由21％提高到35％，P＜0.05)；而不同类型细胞、不同个体、不同年龄、饥饿与否、冷冻与否对异种克隆牦牛早期囊胚发育影响不显著，这为简化供体细胞的处理提供了依据。 4 通过将羚牛和牦牛的异种克隆发育情况、囊胚总细胞数和克隆牛进行比较发现：(1)异种克隆羚牛胚胎在体外可以发育到囊胚阶段，囊胚率为5％左右：(2)利用相同的克隆技术，同种克隆牛、异种克隆牦牛、异种克隆羚牛之间克隆效率差异显著(48％；28％；5％，P＜0.01) (3)三种克隆囊胚的细胞数无差异。这些结果说明牛卵母细胞可以支持羚牛、牦牛、牛成纤维细胞核进行重编程，但是囊胚发育率种内较异种克隆高(牛高于牦牛、羚牛)，物种间距离越近克隆效率越高(牦牛高于羚牛)。 5 由于异种克隆所用的卵母细胞不是同一物种，有必要对克隆后囊胚的核，线粒体遗传物质进行鉴定。本实验对异种克隆牦牛和羚牛囊胚鉴定结果表明：囊胚核基因组来自牦牛和羚牛供核中国农业大学博士学位论文中文摘要细胞，说明本实验获得的囊胚是真正异种体细胞克隆的结果;囊胚线粒体以牛卵母细胞线粒体为主，耗牛或羚牛供体细胞线粒体含量甚微。 6异种克隆耗牛胚胎进行移植:共移植耗牛胚胎185枚，用受体牛60头，其中有近33头的受体牛返情期延长，移植后第60天直肠检查确认有3头怀孕。7个月后，1头牛妊娠仍然正常，为通过异种克隆的方法保护珍稀动物提供了依据。 7目前在较近的物种之间进行异种体细胞克隆虽然已有成功的报道，但是多数报道的异种体细胞克隆都发育到囊胚阶段，最多妊娠几个月。本实验在异种克隆耗牛实验中也发现了这一问题。为了较全面的探索其基因方面的原因，则需要对大量的候选基因进行分析。而利用传统的RT一PCR方法从单个卵母细胞或胚胎中获得的RNA量不足以分析大量的基因。本研究利用并优化了一套全新的扩增细胞中整体cDNA的方法，使微量材料进行大量的基因表达分析成为现实。通过该方法，本实验检测了6个重要基因在耗牛体细胞和牛卵母细胞和耗牛异种体细胞克隆胚胎的表达情况;同时对耗牛异种体细胞克隆囊胚的内细胞团/总细胞的比例进行了检测。结果表明耗牛体细胞功能基因波形蛋白基因和胶原蛋白基因只在耗牛细胞中检测到，说明耗牛体细胞的基因表达得到了重编程。发现cx43和乃油乙夕这两个对细胞和胚胎重要的基因在耗牛细胞和胚胎中均表达;两个对胚胎发育重要的基因人吸”hZ和116不在供体细胞中表达，在胚胎中有表达，但其起始表达的时期异常。耗牛异种体细胞克隆囊胚的内细胞比例显著高于牛人工受精囊胚。由此可推测，滋养层细胞比例过少和胚胎发育重要的基因表达起始时期异常可能与异种体细胞克隆胚胎妊娠困难和流产率高有关。 8在此次研究中发现，作为干细胞标志的Ocl4基因在牛和耗牛体外培养的细胞系中也有大量表达。利用原位杂交技术检测发现Ocl4在牛卵母细胞、牛克隆和IVF胚胎、异种克隆耗牛胚胎、牛体外培养的细胞系和耗牛体外培养的细胞系中均有表达。说明Oct4基因在牛和耗牛中并非只在干细胞中表达，推测该基因可能具有种属特异性。更多还原

【Abstract】 Interspecies cloning might be used as an effective method to conserve endangered species and to support the study of nuclear-cytoplasm interaction. In this study, we describe the development of yak-bovine embryos (or takin-bovine embryos) in vitro produced by fusing yak (or takin) ear fibroblasts with enucleated bovine oocytes, and blastocyst-stage yak-bovine embryos transferred into bovine surrogate mothers established pregnancies, One of which would produce an apparently normal yak in next month.1 We established two takin somatic cell lines and nineteen yak somatic cell lines from the donor animals coming from the Beijing Animal Park, Qinhuangdao Wild Animal Park and Qinghai province. They respectively belonged to the ear fibroblast cell lines, cumulus cells and oviduct cells lines. We found cell lines were normal after the tissues had stayed at 0℃ for 48 hours. These results made for collecting the tissue from endangered animal at the field2 Somatic cell nuclear transfer: we firstly established our enucleated methods by combining H33342 staining method and blind sucked method. Secondly, the parthenogenetic activation of bovine oocytes with chemical stimulation was investigated. The activation rate of the complex chemical activation was higher than the single chemical ones.The complex of A23187 + 6-DMAP got the best result (cleaved 83.6%). At last, we compared the development of the bovine parthenogenetic blastocysts cultured in M199 and CRlaa and found bovine embryos was suitable to be cultured in CR1aa.3 Interspecies cloning yak: we got higher blastocysts development in vitro (35%) by fusing these yak cells with enucleated bovine oocytes. The results suggest that oocytes were the basis for cloning success. When the maturated rate was lower than 40%, the induced blastocyst rate was lower (3% versus 25%, P<0.01). Further, delaying activation following nuclear transfer showed the potential for greatly improving embryo development in vitro. The yak blastocyst development rates increased from 21% to 35% by increasing the activation delayed period from 0.5hr to 1.5hr. No difference was observed among the various sources and the various preparations of donor cells.4 We also compared the blastocyst development of takin-bovine embryos with yak-bovine and bovine-bovine embryos and compared the cell numbers of the blastocyst. Our results indicated that: (1) takin-bovine cloned embryos could develop to the blastocyst stage in vitro (5%), (2) using the same nuclear transfer protocol, development efficiency was significantly different between bovine-bovine cloning, yak-bovine, and takin-bovine cloning (48% versus 28% versus 5%, P<0.01), and (3) cell numbers in the blastocysts of the three species of embryos were not significantly different. These results suggested that the bovine oocytes could reprogram the takin, yak, and bovine fibroblast nuclei. However, the development efficiency of intra-species cloning tended to be higher than inter-species cloning; the closer the species of the donor cell was to the recipient oocyte (yak versus takin), the greater the blastocyst development in vitro.5 Testing the genomic and mitochondrial DNA of yak (or takin) blastocysts: blastocyst genomic DNA only came from the yak (or takin) donor cells while the mitochondria DNA came mainly from bovine oocyte. These suggested the blastocysts come from interspecies cloning.6 Yak-bovine blastocysts transfer: 55 percent surrogate bovine delayed estrus, three of sixty surrogate bovines get pregnancy at 60th day. Now one continues gestation longer than 210 days.7 Although inter-species somatic nuclear transfer (SNT) embryos of evolutionary close species can develop to term, our results fould yak-bovine blastocyst got low pregnancy in surrogate bovine. It was important to analyze a wide variety of genes in individual cloned embryos. But using conventional RT-PCR strategies permitted analysis of only a few genes in one embryo. In this study, a global RT-PCR strategy has been used, allowing the analysis of almost hundre更多还原