摘要
首例体细胞克隆猪已出生十多年,但核移植效率一直很低,困扰着克隆猪的应用。体细胞核移植通常采用分化的体细胞和成熟的卵母细胞,供体细胞和卵母细胞的质量直接决定着后期克隆胚胎发育。目前认为克隆效率低的主要原因是供体细胞核未被完全重编程,主要体现在基因组高甲基化水平。体细胞核移植后细胞核表观遗传修饰重建是影响到克隆胚胎能否发育到期的关键因素,因此通过调控克隆胚胎的表观遗传修饰可能会提高核移植效率。本研究拟通过对胞质成熟和供体细胞在猪克隆胚胎中发挥的作用进行研究,探索胞质成熟和供体细胞对核移植胚胎发育的影响,同时利用表观修饰药物和扰低DNA甲基转移酶1探讨和阐明表观遗传修饰状态以及关键基因的表达水平在克隆胚胎重编程过程中的作用,希望为提高猪体细胞核移植效率提供理论依据。主要研究结果如下：
(1)在卵母细胞体外成熟中,探讨了胞质成熟对核移植胚胎发育的影响,结果表明,卵母细胞体外培养33h即可达到核成熟,之后到42h之间的时间为胞质成熟,胞质成熟能够显著提高猪克隆胚胎的发育能力(P<0.05),这表明充分的胞质成熟是核移植胚胎发育的关键。
(2)供体细胞状态影响着核移植胚胎发育,对供体细胞种类、性别和甲基化程度进行分析,结果表明：相对于卵丘细胞和成体成纤维细胞,胎儿成纤维细胞更适合于体细胞核移植；与雄性胎儿成纤维细胞相对,雌性胎儿成纤维细作为供体细胞时核移植胚胎发育能力差异不显著,而雌性胎儿成纤维细更适合于X染色体失活的机理研究；5-aza-dC或5-methyl-dC能够显著降低或提高胎儿成纤维细胞的甲基化水平(P<0.05),但并不利于核移植效率的提高。这表明雌性胎儿成纤维细胞更适合作为体细胞核移植的供体细胞,而改变其甲基化水平不利于体细胞重编程。
(3)利用5-aza-dC对供体细胞和核移植胚胎进行处理,分析了处理后对细胞增殖、胚胎发育、核重塑及基因表达等的影响,结果表明：5-aza-dC对供体细胞增殖具有抑制作用,能够显著降低DNA甲基转移酶1和3a和提高Igf2和Xist在供体细胞中的表达水平(P<0.05),处理供体细胞后不利于核移植胚胎的发育(主要表现为核重塑能力差、胚胎卵裂率低以及DNA甲基转移酶基因和多能性基因表达水平未得到改善)；5-aza-dC处理核移植胚胎能够显著增强胚胎发育能力(P<0.05),其中25nmol处理24h效果最佳,5-aza-dC处理核移植胚胎后显著加快了供体细胞核基因的重编程,促进了类原核形成和早期卵裂,改善了DNA甲基转移酶基因、印记基因、多能性基因以及去甲基化相关基因的表达水平,使其接近于体外受精胚胎表达模式；5-aza-dC同时处理供体细胞和核移植胚胎,发育能力得到更显著的提高(P<0.05),这可能是双重处理更有利于供体核的重编程。
(4)利用TSA对核移植胚胎进行处理,观察了处理后体内外克隆胚胎的发育能力,同时对TSA处理期间基因表达的变化进行了分析,结果表明：TSA能够显著增加核移植胚胎的体外发育率；进行胚胎移植后,相对于正常核移植胚胎,处理组提高了克隆胚胎妊娠率和出生率,并能有效的防止大舌头病和死胎的发生；TSA处理后显著加快了供体细胞核基因的重编程和改善了DNA甲基转移酶基因的表达(P<0.05)。这表明TSA处理能够增强供体核的重塑和纠正胚胎发育相关基因的表达,从而增强体内外胚胎发育能力。
(5)利用RNAi扰低Dnmt1或许能够改善DNA的去甲基化从而提高核移植胚胎的发育能力,对RNAi体系、Dnmt1扰低后的卵母细胞成熟和核移植胚胎发育能力进行了分析,结果表明,壁颗粒细胞共培养可以增强裸卯成熟质量,为干扰体系奠定了了基础; Dnmt1扰低后不影响卵母细胞的成熟；Dnmt1扰低后显著降低了核移植胚胎的发育(P<0.05),多数胚胎阻滞在4和8细胞。这表明Dnmt1在核移植胚胎早期发育过程中起着重要作用,适当的表达水平才是决定胚胎发育的因素。
The first cloned pigs have been born over ten years, however, the efficiency of nuclear transfer has been low, limiting the application of cloned pigs. Differentiated somatic cells and matured oocytes were used in somatic cell nuclear transfer, thus their qualities directly determine the developmental competence of cloned embryos. At present, the main reason for low cloning efficiency is that donor nucleus is not completely reprogrammed, showing genome highly methylated. Epigenetic modification in somatic cell nuclear transfer is the key factor affecting on the development of cloned embryos, thus regulation of epigenetic modification in cloned embryos may improve the cloning efficiency. This study aimed to analyze the role of cytoplasmic maturation and donor cells in somatic cell nuclear transfer in order to evaluate the effect of cytoplasmic maturation and donor cells on the development of cloned embryos and apply epigenetic modification drugs and knockdown of DNA methyltransferase1to explore and elucidate the molecular mechanism of key genes in the somatic cell nuclear reprogramming, and thus provides a theoretical basis to improve the efficiency of porcine somatic cell nuclear transfer. The main results are as follows.
(1) During the progress of oocytes in vitro maturation, we analyzed the effect of cytoplasmic maturation on the developmental competence of cloned embryos, and the results showed that oocytes cultured for33h have achieved nuclear maturation and the time between33h and42h was cytoplasmic maturation. The results also showed cytoplasmic maturation could significantly enhance the developmental competence of porcine cloned embryos (p<0.05), though cytoplasmic maturation was not conducive to remove nuclear materials. These results indicate that full cytoplasmic maturation is the key factor in the development of cloned embryos.
(2) Donor cell is a determining factor in somatic cell nuclear transfer, thus the types, sexes and methylation levels of donor cells were analyzed. It was shown that compared with cumulus cells and adult fibroblasts, fetal fibroblasts was more suitable for somatic cell nuclear transfer in term of the fusion rate and cell proliferation. As for sexes, there were not significant differences in the cloned embryo development between male and female fetal fibroblasts, while female fetal fibroblasts are more suitable for the research on the mechanism of X chromosome inactivation. The results also showed that5-aza-dC or5-methyl-dC could significantly decrease or increase the methylation levels of fetal fibroblast cells (p<0.05), however, the changes of methylation levels could not enhance the cloning efficiency. It indicates that female fetal fibroblasts are more suitable as donor cells, while th change in the methylation level is not conducive to improve nuclear reprogramming.
(3) In order to enhance the cloned embryo development,5-aza-dC was applied to treat donor cells and cloned embryos, and cell proliferation, cloned embryo development, nuclear remodeling and gene expression were studied. When donor cells were treated, the cell proliferation was inhibited, and the expression levels of DNA methyltransferase1and3a were significantly reduced while those of Igf2and Xist significantly increased (p<0.05), furthermore, the development of cloned embryos was not enhanced, showing that the ability of nuclear remodeling and cleavage rates were low and the expression levels of DNA methyltransferase and pluripotent genes did not improve. When cloned embryos were treated, the development competence was significantly enhance (p<0.05), and the treatment manner with25nmol for24h was best. Treatment cloned embryos significantly (p<0.05) accelerated the reprogramming of donor cell nuclear genes, promoted the pronucleus-like formation and early cleavage, and improved the expression levels of DNA methyltransferase, imprinting, pluripotent and demethylation-related genes, similar to those in IVF embryos. It was surprised that when both donor cells and cloned embryos were treated, the developmental competence was more significantly enhanced (p<0.05), indicating that double treatment is more suitable for the reprogramming of donor nuclei.
(4) We analyzed the developmental competence of cloned embryos in vitro and in vivo after treatment cloned embryos with TSA and gene expression levels during TSA treatment, and found that TSA could significantly enhance the developmental competence of cloned embryos in vitro (p<0.05), Furthermore, after embryo transfer, compared with normal cloned embryos, TSA treatment improved pregnancy and birth rates, and prevented the occurrence of large tongue disease and stillbirth effectively. After TSA treatment, the reprogramming of donor cell nuclear and DNA methyltransferase genes was significantly accelerated (p<0.05). These results above indicates that TSA treatment could enhance the donor nuclear remodeling and correct embryo development related gene expression, and thus enhance the developmental competence of cloned embryos in vitro and in vivo.
(5) Knockdown of Dnmtlby RNA interference may lead to effectiveDNA demethylation, and thus improve the developmental competence of cloned embryos. We analyzed the state of RNAi system, and oocyte maturation and cloned embryo development after Dnmtl interference, and found that RNAi system was steadily set up, as mural granulosa cells could enhance the maturation quality of deduded oocytes and the maturation rate of oocytes after Dnmtl knockdown did not change, however, it was surprised to find that the developmental competence of cloned embryos derived from oocytes of Dnmtl knockdown significantly decreased (p<0.05), and many cloned embryos were arrested at4and8cell stage. These results suggests that Dnmtl plays an important role in the early developmental process of cloned embryos, and appropriate expression level is the determining factor for embryo development.
引文
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