治疗性克隆

治疗性克隆是利用核移植技术将病人的体细胞核移植到核的卵母细胞中,使其重编程并发育成囊胚,然后再用胚胎干细胞分离技术从克隆囊胚的ICM分离出多能胚胎干细胞（ES）。这种干细胞在遗传学上和病人完全一致,再定向诱导其分化成病人所需要的体细胞进行移植,以取代和修复患者已丧失功能的细胞、组织或器官,而达到完全治愈。治疗性克隆不仅解决移植物与受者间的免疫排斥反应问题,而且可以解决移植物的来源问题。     

治疗性克隆研究进展

核移植来源的胚胎干细胞 (ES) 细胞在动物疾病模型上的成功治疗作用以及核移植来源的患者特异的ES细胞研究进展有望将这一技术即治疗性克隆应用于人类疾病治疗.本文对治疗性克隆的研究进展、治疗性克隆研究中存在的问题以及治疗性克隆的应用前景作一综述和讨论.     

治疗性克隆

治疗性克隆是利用核移植技术将病人的体细胞核移植到去核的卵母细胞中 ,使其重编程并发育成囊胚 ,然后再用胚胎干细胞分离技术从克隆囊胚的ICM分离出多能胚胎干细胞 (ES)。这种干细胞在遗传学上和病人完全一致 ,再定向诱导其分化成病人所需要的体细胞进行移植 ,以取代和修复患者已丧失功能的细胞、组织或器官 ,而达到完全治愈。治疗性克隆不仅解决移植物与受者间的免疫排斥反应问题 ,而且可以解决移植物的来源问题。     

核移植与治疗性克隆

核移植与治疗性克隆在畜牧业生产以及生物医学上具有广阔和诱人的应用前景。文章分析指出卵母细胞质量与供核细胞重新编程是影响体细胞核移植效率及克隆动物异常的主要因素,阐述了治疗性克隆所面临的一些基本问题及出路：治疗性克隆以核移植技术为基础,核移植所面临的一些问题也直接影响着治疗性克隆的临床应用;核移植胚胎干细胞分离培养效率的高低以及向重要功能细胞定向分化是治疗性克隆的前提;成体干细胞可用于一些重大疾病的治疗,但不能完全替代克隆性治疗;伦理问题也阻碍治疗性克隆的发展。核移植及治疗性克隆技术要想尽快更好地应用于临床和造福于人类,需要不断完善各技术环节和加强一些基础理论的研究。Abstract: Nuclear transfer and therapeutic cloning have widespread and attractive prospects in animal agriculture and biomedical applications. We reviewed that the quality of oocytes and nuclear reprogramming of somatic donor cells were the main reasons of the common abnormalities in cloned animals and the low efficiency of cloning and showed the problems and outlets in therapeutic cloning, such as some basic problems in nuclear transfer affected clinical applications of therapeutic cloning. Study on isolation and culture of nuclear transfer embryonic stem (ntES) cells and specific differentiation of ntES cells into important functional cells should be emphasized and could enhance the efficiency. Adult stem cells could help to cure some great diseases, but could not replace therapeutic cloning. Ethics also impeded the development of therapeutic cloning. It is necessary to improve many techniques and reinforce the research of some basic theories, then somatic nuclear transfer and therapeutic cloning may apply to agriculture reproduction and benefit to human life better.     

核移植胚胎干细胞的研究及其应用前景

随着核移植技术和干细胞技术的逐渐成熟,目前已获得牛、小鼠核移植胚胎干细胞,以及人 - 兔异种间核移植胚胎干细胞,这些细胞在体外可分化成多种细胞形态 . 已经进行的实验性动物克隆性治疗,显示了诱人的潜力,但人核移植胚胎干细胞研究还面临着许多问题,如建系效率低、卵母细胞来源有限以及伦理学和安全性问题等 . 长远地看,随着克隆效率的提高,在道德与法律之间达成共识,核移植胚胎干细胞必将造福人类 .     

核移植技术克隆动物胚胎研究的现状及展望

胚胎克隆,即建立胚胎的无性繁殖系,最有效的方法是通过用核移植技术将一枚多细胞胚胎的每一细胞核移植到一个去核的、激活的成熟卵母细胞中,重新构成新的胚胎,重组胚胎移植后,发育成与供体胚胎基因型相同的后代。Spemann 最先提出将多细胞胚的每一细胞核分别转移到去核的卵母细胞中这一想法。Brig-gs 和 King 沿用 Spemann 的想法及实验技术,     

人-兔异种核移植构建克隆胚的实验研究

“治疗性克隆”是人类最关注的课题之一,而人体细胞核移植是治疗性克隆的基础和前提。异种核移植的方法虽已被引入人体细胞克隆胚的构建,但供体细胞的类型、培养代数及准备方法与其效率之间的关系尚有待探讨。本实验以不同培养代数和不同准备方法的人卵丘细胞、皮肤成纤维细胞和软骨细胞为供体构建了克隆胚,对其发育情况的比较表明,以卵丘细胞为供体时重构胚的体外发育率高于其余二者,差异显著（P〈0．05）;不同培养代数的成纤维细胞克隆胚和不同冷藏天数供体细胞克隆胚体外发育率无明显差异。此外,本实验还尝试用荧光原位杂交法检测所构建的异种克隆胚核遗传物质的来源,结果显示来自人体细胞。本研究表明,人一兔异种核移植构建克隆胚切实可行;体细胞的类型与核移植效率相关;供体细胞的体外培养传代对克隆胚的发育并无影响;而冷藏是一种简便有效的供体细胞准备方法;此外,用FISH方法对重构胚进行核遗传物质的鉴定切实可行。     

核移植与诱导多能干细胞技术在体细胞重编程研究中的应用

体细胞重编程是指分化的体细胞在特定的条件下被逆转后恢复到多能性或全能性状态,或者形成多能干细胞系,或者形成早期胚胎然后发育成一个新的个体的过程。诱导体细胞重编程的方法有许多,如核移植（nuclear transfer,NT）、细胞融合、细胞培养和通过导入特定因子获得诱导多能干（induced pluripotent stem,iPS）细胞的方法等。其中核移植和iPS技术是到目前为止诱导体细胞为多能干细胞最为完全、最具有运用于临床再生医学潜能的方法。然而,它们的效率都很低,机制也不清楚,如何将两个方法结合在一起,提高重编程的效率,揭示重编程的机制,进而促进其在患者特异性治疗中的运用将是下阶段的努力方向。     

哺乳动物体细胞核移植研究进展

体细胞核移植技术已经在基础研究领域与产业化应用领域体现出了重要的价值,因而体细胞核移植技术及其相关研究已经成为了生物领域的持续性研究热点,但是围绕体细胞核移植技术仍然存在许多质疑,其中最主要的就是体细胞核移植的效率较低。尽管如此,体细胞核移植研究仍然在近年来取得了令人瞩目的成就,包括小鼠与恒河猴核移植胚胎干细胞系的建立。该文就体细胞核移植的研究历史与进展进行简要的论述,同时针对体细胞核移植研究中的细胞重编程与治疗性克隆研究中的发展与问题进行剖析,希望能够积极推动治疗性克隆的研究进展,加速核移植与干细胞技术在产业化领域中的应用。     

体细胞起源的人胚胎干细胞

根据治疗性克隆假设,可以通过体细胞核移植技术获得与病人具同样基因型的细胞或组织。这样起源的细胞或组织植回病人将不会引起免疫排斥反应。本研究将5岁、42岁、52岁和60岁4个不同年龄的人体细胞核植入去核的兔卵母细胞中重新启动,发育至囊胚,并分离人胚胎干细胞。研究结果提示,年龄不影响体细胞被重新启动的效率。经过核型分析,同源染色体分析,原位杂交,PCR和免疫组化染色等多种鉴定,ntES细胞具有人染色体。ntES细胞可以长期增殖并保持不分化状态,也可以形成类胚体并分化出包括神经和肌肉在内的多种细胞类型。由类胚体诱导生成的混合细胞群体表达所有三个胚层(外、中、内胚层)细胞类型标记,说明ntES细胞具有分化成所有三个胚层的潜力。因此,从人体细胞核获得的ntES细胞与普通人胚胎干细胞一样具有向多种细胞类型分化的能力。     

Cloned mice and embryonic stem cell establishment from adult somatic cell

Cloning methods are now well described and becoming routine. Yet the frequency at which cloned offspring are produced remains below 2% irrespective of nucleus donor species or cell type. Especially in the mouse, few laboratories can make clones from adult somatic cells, and most mouse strains never succeed to produce cloned mice. On the other hand, nuclear transfer can be used to generate embryonic stem (ntES) cell lines from a patient's own somatic cells. We have shown that ntES cells can be generated relatively easily from a variety of mouse genotypes and cell types of both sexes, even though it may be more difficult to generate clones directly. Several reports have already demonstrated that ntES cells can be used in regenerative medicine in order to rescue immune deficient or infertile phenotypes. However, it is unclear whether ntES cells are identical to fertilized embryonic stem (ES) cells. In general, ntES cell techniques are expected to be applicable to regenerative medicine, however, these techniques can also be used for the preservation of the genetic resources of mouse strains instead of preserving such resources in embryos, oocytes or spermatozoa. This review seeks to describe the phenotype, application, and possible abnormalities of cloned mice and ntES cell lines.     

细胞核重编程对克隆的影响

细胞核重编程是哺乳动物正常受精胚胎和克隆胚胎发育过程中的一个重要特性,主要是对表观遗传学特征进行重新编写,包括染色质重塑、组蛋白修饰、DNA甲基化、印记基因表达、X染色体失活等表观遗传修饰的改变。通过细胞核重编程,首先,受精卵和克隆胚胎的供体核停止其特有的基因表达程序,恢复为全能状态的基因表达程序;然后,受精胚胎和克隆胚胎的细胞再从全能状态重新进入分化状态,最终形成各种组织和器官。近年来,不少研究表明,克隆胚胎的细胞核重编程存在不同程度的表观遗传修饰异常,可能对克隆及其农业和医学应用有着重要影响。本文就正常和克隆胚胎细胞核重编程的研究进展以及克隆胚胎的细胞核重编程异常对克隆的影响作一综述,并对目前有关治疗性克隆前景的不同看法进行了讨论。     

Establishment of customized mouse stem cell lines by sequential nuclear transfer

Therapeutic cloning, whereby embryonic stem cells （ESCs） are derived from nuclear transfer （NT） embryos, may play a major role in the new era of regenerative medicine. In this study we established forty nuclear transfer-ESC （NTESC） lines that were derived from NT embryos of different donor cell types or passages. We found that NT-ESCs were capable of forming embryoid bodies. In addition, NT-ESCs expressed pluripotency stem cell markers in vitro and could differentiate into embryonic tissues in vivo. NT embryos from early passage RI donor cells were able to form full term developed pups, whereas those from late passage RI ES donor cells lost the potential for reprogramming that is essential for live birth. We subsequently established sequential NT-RI-ESC lines that were developed from NT blastocyst of late passage R 1 ESC donors. However, these NT-R I-ESC lines, when used as nuclear transfer donors at their early passages, failed to result in live pups. This indicates that the therapeutic cloning process using sequential NT-ESCs may not rescue the developmental deficiencies that resided in previous donor generations.     

体细胞核移植后核重编程的影响因素

近年来,人类核移植胚胎干细胞建系成为一项炙手可热的研究,用再生医学的理念治疗退行性疾病及器官移植为这一研究带来无穷的魅力和生命力;但是核重编程仍是核移植技术的瓶颈,制约了重构胚胎干细胞的研究。核重编程是指供体细胞核移入卵母细胞后必须停止本身的基因表达程序并恢复为胚胎发育所必需的特定的胚胎表达程序。只有供核发生完全重编程,重构胚胎才能正常发育。核重编程与供核者的年龄,供核细胞的组织来源、分化状态、细胞周期、传代次数,供核的表遗传标记以及供卵者的年龄、卵子的成熟度等因素有关。一般来说,颗粒细胞作为核供体最易被核重编程。供核者为胎体或新生体,供核细胞处于低分化状态或已传数代,供核细胞经过去表遗传标记处理,供卵者性成熟且年龄轻、卵子核与胞浆都成熟等均为有利于核重编程的因素。重构胚胎的培养方法对核重编程也至关重要,目前主张使用序贯培养及体细胞化培养。创造各种适于核重编程的条件有利于从更高的起点开展核移植胚胎干细胞研究,提高重构胚胎干细胞建系效率。     

Embryonic stem cells generated by nuclear transfer of human somatic nuclei into rabbit oocytes

To solve the problem of immune incompatibility, nuclear transplantation has been envisaged as a means to produce cells or tissues for human autologous transplantation. Here we have derived embryonic stem cells by the transfer of human somatic nuclei into rabbit oocytes. The number of blastocysts that developed from the fused nuclear transfer was comparable among nuclear donors at ages of 5, 42, 52 and fi0 years, and nuclear transfer (NT) embryonic stem cells (ntES cells) were subsequently derived from each of the four age groups. These results suggest that human somatic nuclei can form ntES cells independent of the age of thedonor. The derived ntES cells are human based on karyotype, isogenicity, in situ hybridization, PCR and immunocytochemistry with probes that distinguish between the various species. The ntES ceils maintainthe capability of sustained growth in an undifferen tiated state, and form embryoid bodies, which, on furtherinduction, give rise to cell types such as neuron and muscle, as well as mixed cell populations that expressmarkers representative of all three germ layers. Thus, ntES cells derived from human somatic cells by NTto rabbit eggs retain phenotypes similar to those of conventional human ES ceils, including the ability toundergo multilineage cellular differentiation.