牛体细胞核移植中的异常重编程及提高核移植效率的措施

体细胞核移植技术具有重要的理论和实践价值,但是其成功率依然很低。目前普遍认为,核移植效率低下的原因是体细胞重编程不完全,即体细胞的表观印记未被完全去除,未能建立正确的胚胎基因表达模式,进而出现各种发育阻滞和异常。论文综述了近年来对牛体细胞核移植重编程机理的探索以及提高牛体细胞核移植效率的措施。     

哺乳动物体细胞核移植的DNA甲基化重编程研究进展

DNA甲基化是基因组主要的表观遗传修饰方式之一.核移植重构胚在对供体细胞基因组进行甲基化重编程过程中会出现异常的甲基化模式,而异常的甲基化重编程是导致克隆胚早期死亡及克隆动物发育畸形的主要原因.论文针对体细胞克隆动物基因组DNA的甲基化模式、造成克隆胚胎甲基化异常的原因及异常甲基化对重构胚胎发育的影响等进行了综述.深入研究核移植重构胚甲基化重编程的机制,有助于完善核移植技术,提高克隆效率,使其更好地应用于基础研究和生产实践.     

表观遗传修饰影响哺乳动物体细胞核移植效率的研究进展

表观遗传修饰是一种不依赖于DNA序列变化的可逆、可遗传修饰，在哺乳动物胚胎发育的整个阶段均可发生，是影响哺乳动物体细胞核移植效率的主要因素之一。其中，DNA甲基化、组蛋白的动态修饰、X染色体失活、端粒与端粒酶活性变化作为常见的表观遗传修饰类型，任一修饰形式的异常都会影响基因的表达，引发体细胞重编程错误导致核移植效率降低。近年来，随着体细胞核移植技术研究的不断深入，表观遗传修饰影响体细胞核移植效率的关键作用机制日益明确。本文通过综述不同类型的表观遗传修饰影响哺乳动物体细胞核移植效率的研究进展，以期在表观遗传修饰层面为提高哺乳动物体细胞核移植效率提供新思路。     

小鼠雌性多能干细胞X染色体的表观遗传状态研究进展

雌性多能干细胞(iPS细胞)多能性状态的获得伴随着X染色体的表观修饰重编程。分化终末状态的雌性体细胞中有1条X染色体发生异染色质化而导致其失活。在体细胞诱导多能性干细胞的过程中,失活的X染色体重新活化。在重编程过程中,多能因子和X染色体失活中心的非编码基因的联系紧密。文章主要从分子水平上讨论多能干细胞X染色体表观修饰的相关研究进展。小鼠胚胎干细胞(ES细胞)是标准的多能性基态。X染色体上非编码RNA的表达可能是一个评价iP S表观遗传状态的标记,相关研究可以为细胞治疗的临床应用提供重要依据。     

哺乳动物体细胞核移植胚胎发育中表观重编程的研究进展

体细胞核移植（somatic cell nuclear transfer,SCNT）是一种能将已分化的体细胞重编程为全能胚胎的繁殖生物技术,在良种扩繁、濒危物种保护和治疗性克隆等方面有着广泛的应用前景,但极低的克隆效率、克隆动物胎盘异常、出生后胎儿畸形等严重限制了该技术的实际应用。造成克隆效率低和胚胎发育异常的主要原因是供体核表观遗传重编程错误或不完全。1958年,将非洲爪蟾（Xenopus laevis）幼体肠细胞核移入去核卵母细胞,获得了第1例SCNT动物个体;1986年,通过电融合1个卵裂球与去核卵母细胞成功获得了3只存活的羔羊;1997年,将成年母羊的乳腺上皮细胞与去核卵细胞电融合,获得首个SCNT哺乳动物"多利",开启了克隆时代,目前牛、小鼠、山羊、猪、欧洲盘羊、家兔、家猫、马、大鼠、骡子、狗、雪貂、狼、水牛、红鹿、单峰骆驼、食蟹猴等相继成功克隆,其中最引人瞩目的是2018年食蟹猴的成功克隆。作者通过将SCNT胚胎与受精胚胎的发育进行对比,阐述了SCNT过程中DNA甲基化、组蛋白修饰、基因组印迹、染色体状态等的重编程过程和缺陷,并从表观修饰剂、组蛋白去甲基化酶、抑制Xist表达、补充鱼精蛋白和精子RNA方面探讨单独或联合消除表观遗传重编程障碍对克隆效率的影响。随着低样本量测序技术的发展和完善,人们能够在SCNT胚胎中检测到更详细的全基因组表观遗传修饰图谱,进一步揭示SCNT胚胎表观遗传重编程中的缺陷,为提高克隆效率提供了线索。通过上述内容的阐述,希望为后续开发联合消除多种表观遗传障碍而提高克隆效率的策略和思路。     

基因组表观重编程对体细胞核移植成功率的影响

基因组表观重编程缺陷是影响体细胞核移植效率的主要因素,本文讨论了表观重编程的两大主要机制——DNA甲基化及组蛋白修饰及其对体细胞核移植重构胚胎发育的影响,并综述了几种促进核重编程的方法。     

体细胞克隆中核的重编程

尽管体细胞克隆在绵羊、牛、小鼠、猪、山羊、兔、猫、大鼠和骡子等物种中都获得了成功，但却未能得到狗和猕猴的克隆个体，而且克隆效率非常低，克隆效率低使体细胞克隆技术在科研和生物技术等方面的应用受到限制．供体核移入去核的卵细胞后，必须经过表观遗传修饰的重编程，回到胚胎开始发育的全能状态。目前认为：供体核的不完全重编程是导致克隆效率低的主要原因。本文从DNA甲基化、组蛋白乙酰化、X染色体失活、端粒、印记基因以及其他发育相关基因的表达几个方面来探讨影响克隆效率的因素。     

WT1基因组蛋白乙酰化修饰对细胞核重编程的影响

利用体细胞移植技术获得克隆动物的成功是几十年来生命科学领域取得的重大突破之一,这项技术引起了社会的广泛关注。然而,由于哺乳动物克隆效率低下,且克隆后代发育异常等问题,已成为目前制约动物克隆技术发展和应用的瓶颈。克隆动物中经常出现后代过大综合征（LOS）,该病导致克隆动物早产、难产和易夭折。LOS类似于人的伯-伟综合征（BWS）,BWS也称为Wlims瘤,表现为巨舌、内脏肿大等症状。研究发现BWS的发病机理与WT1基因（Wilms’tumor 1gene）异常表达有关。本文对体细胞核重编程和表观遗传学调控细胞重编程的研究进展进行综述,并对WT1基因组蛋白乙酰化修饰与体细胞重编程之间的联系进行简要介绍,以期为生命科学领域的进一步探索与研究提供借鉴。     

鸡体细胞诱导重编程早期的糖代谢变化研究

为研究鸡体细胞诱导重编程早期的糖代谢方式的变化,试验采用OCT4、SOX2、NANOG和LIN28A(OSNL)四因子诱导体系将鸡胚成纤维细胞(Chicken embryo fibroblasts,CEF)重编程为诱导多能干细胞(Induced pluripotent stem cells,iPS),并利用碱性磷酸酶染色、阶段特异性胚胎抗原1(Stage-specific embyronic antigen-1,SSEA-1)免疫荧光染色、体外诱导分化及多能性基因表达检测等对iPS进行鉴定。通过检测重编程过程中糖代谢相关基因表达及酶活性的变化,并对葡萄糖摄取量、乳酸产生量及线粒体膜电位检测等研究鸡体细胞诱导重编程早期的糖代谢变化。结果显示,鸡CEF诱导重编程形成的iPS呈碱性磷酸酶染色阳性,表达SSEA-1蛋白,体外分化形成类胚体且表达多能性标记基因。同时重编程过程中氧化磷酸化基因表达下调而糖酵解相关基因表达上调,糖酵解关键酶活性均增强,且iPS的葡萄糖吸收量及乳酸产生量增加,而线粒体膜电位则下降。结果表明,OSNL四因子体系将鸡CEF诱导重编程形成iPS的过程中,细胞的主要糖代谢方式从氧化磷酸化转变为糖酵解,而糖酵解的激活可能会进一步促进iPS的形成。     

小分子化合物促进体细胞重编程为多能干细胞的研究进展

诱导多能干细胞(induced pluripotent stem cells,iP SCs)是指通过转入外源转录因子将体细胞重编程为具有胚胎干细胞(embryo stem cells,ESCs)特性与功能的一种细胞。iP SCs在细胞治疗、体外疾病模型建立与机理研究、药物发现及评价等方面有着巨大的潜在应用价值。近年来,重编程技术发展快速,然而仍处在了解细胞重编程机制的早期阶段。小分子化合物作为一种简单的操作工具,可以调控细胞的不同信号通路、表观遗传及新陈代谢等。在重编程过程中,它能够显著提高iP SCs的重编程效率,可以替代相关转录因子进行重编程,也可以促进初始态多能性(Na6ve)的转化。文章旨在总结近几年来小分子化合物在诱导体细胞重编程中的作用,为进一步完善iP SCs重编程技术提供参考。     

Establishment,Differentiation, Electroporation and Nuclear Transfer of Porcine Mesenchymal Stem Cells

The limited success of somatic cell nuclear transfer (SCNT) is largely attributed to defects in epigenetic reprogramming of the donor genome. Donor cell types with distinct potential competence may offer different epigenetic flexibility for subsequent genome reprogramming in SCNT. Stem cells possibly enable their genomes to be more readily reprogrammed than differentiated cells. To improve the efficiency of cloning, porcine mesenchymal stem cells (pMSCs) were isolated and well identified by 6‐channel flow cytometry and differentiation assays and were used as donors in SCNT. Compared with porcine embryonic fibroblasts (pEFs), our results showed that pMSCs markedly enhanced cloned embryo development in terms of cleavage and blastocyst formation (p < 0.05). To enhance the epigenetic flexibility of pMSCs, classical reprogramming factors (RFs) were transfected by electroporation, and we achieved optimization with ectopic expression of RFs in pMSCs. Our results suggest that the epigenetic status of donor cells has an improvement on genome reprogramming, and multipotent pMSCs favoured subsequent embryonic development.     

Inheritance of histone H3 methylation in reprogramming of somatic nuclei following nuclear transfer

Successful cloning requires reprogramming of epigenetic information of the somatic nucleus to an embryonic state. However, the molecular mechanisms regarding epigenetic reprogramming of the somatic chromatin are unclear. Herein, we transferred NIH3T3 cell nuclei into enucleated mouse oocytes and evaluated the histone H3 dimethyl-lysine 4 (H3K4me2) dynamics by immunocytochemistry. A low level of H3K4me2 in the somatic chromatin was maintained in pseudo-pronuclei. Unlike in vitro fertilized (IVF) embryos, the methylation level of nuclear transfer (NT) embryos was significantly increased at the 8-cell stage. NT embryos showed lower H3K4me2 intensity than IVF embryos at the 2-cell stage, which is when the mouse embryonic genome is activated. Moreover, the H3K4me2 signal was weak in the recloned embryos derived from single blastomeres of the NT embryos, whereas it was intense in those from IVF embryos. Two imprinted genes, U2afbp-rs and Xist, were abnormally transcribed in cloned embryos compared with IVF embryos, and this was partly correlated to the H3K4me2 level. Our results suggest that abnormal reprogramming of epigenetic markers such as histone acetylation and methylation may lead to dysregualtion of gene expression in cloned embryos.     

绵羊Sox2基因逆转录病毒载体的构建与检测

为建立利用内源诱导因子诱导绵羊体细胞为多能干细胞(induced pluripotent stem cell,iPS)的方法,对绵羊Sox2基因进行克隆,并与pMXs连接构建逆转录病毒载体,将构建的载体转染293GP细胞以获得假病毒上清,利用假病毒上清侵染绵羊胎儿成纤维细胞以检测细胞中的Sox2表达变化。PCR和酶切鉴定结果显示,成功构建了pMXs-Sox2重组质粒,该质粒具有转染293GP细胞的能力,所获得的假病毒上清侵染绵羊胎儿成纤维细胞后,可诱导细胞表达Sox2基因。本研究为开展绵羊iPS的相关研究提供依据。     

miRNA对干细胞重编程的影响

miRNA在胚胎干细胞(ES)细胞的自我更新及多能性中扮演重要角色,国内外研究结果表明,在体细胞形成诱导性多能干细胞(IPS)的过程中,许多miRNA与Sox2、Oct4和Nanog等调控因子组成调控网络。miRNA在细胞周期、重编程及表型建立过程中也起着重要的调控作用。另外,在IPS细胞形成中,miRNA很有可能代替关键转录因子。     

Cell-free extract from porcine induced pluripotent stem cells can affect porcine somatic cell nuclear reprogramming

Pretreatment of somatic cells with undifferentiated cell extracts, such as embryonic stem cells and mammalian oocytes, is an attractive alternative method for reprogramming control. The properties of induced pluripotent stem cells (iPSCs) are similar to those of embryonic stem cells; however, no studies have reported somatic cell nuclear reprogramming using iPSC extracts. Therefore, this study aimed to evaluate the effects of porcine iPSC extracts treatment on porcine ear fibroblasts and early development of porcine cloned embryos produced from porcine ear skin fibroblasts pretreated with the porcine iPSC extracts. The Chariot^TM reagent system was used to deliver the iPSC extracts into cultured porcine ear skin fibroblasts. The iPSC extracts-treated cells (iPSC-treated cells) were cultured for 3 days and used for analyzing histone modification and somatic cell nuclear transfer. Compared to the results for nontreated cells, the trimethylation status of histone H3 lysine residue 9 (H3K9) in the iPSC-treated cells significantly decreased. The expression of Jmjd2b, the H3K9 trimethylation-specific demethylase gene, significantly increased in the iPSC-treated cells; conversely, the expression of the proapoptotic genes, Bax and p53, significantly decreased. When the iPSC-treated cells were transferred into enucleated porcine oocytes, no differences were observed in blastocyst development and total cell number in blastocysts compared with the results for control cells. However, H3K9 trimethylation of pronuclear-stage-cloned embryos significantly decreased in the iPSC-treated cells. Additionally, Bax and p53 gene expression in the blastocysts was significantly lower in iPSC-treated cells than in control cells. To our knowledge, this study is the first to show that an extracts of porcine iPSCs can affect histone modification and gene expression in porcine ear skin fibroblasts and cloned embryos.