动物异种细胞核移植技术研究进展

作为研究动物胚胎发育过程中核质关系重要手段的细胞核移植技术正在逐步完善。异种细胞核移植技术不仅可用于保护濒危野生动物和珍贵的动物遗传资源,而且在研究核质相互作用和物种之间的进化关系上有着独特的用途。尽管目前同种核移植在许多哺乳动物上都获得成功,但异种核移植研究还刚刚开始,有许多问题亟待解决。本仅就现已发表的少量献,简要综述了动物异种核移植研究的历史与现状、影响因素、面临问题和应用前景。     

哺乳动物体细胞异种核移植胚胎早期发育的研究进展

体细胞异种核移植是指将一个物种的体细胞移植到另一物种的去核卵母细胞中,移入的体细胞核在受体胞质中重编程并发育成新个体的实验方法.该方法为拯救濒危物种和获取灵长类胚胎干细胞提供了可能的途径.但这方面的研究目前还只获得初步的进展,核重编程不完全以及异种胚胎的囊胚率低仍是其面临的主要难点.本文从基因表达、表观重编程、线粒体异质性、核重塑和核移植体系优化等方面入手,介绍近年来哺乳动物体细胞异种核移植的研究进展,并探讨异种重构胚重编程所面临的关键问题和可能获得成功的方法.     

继代核移植能成倍提高来自奶山羊卵胞质体的异种间核移植重构胚的发育率

为了提高异种间核移植重构胚的发育率,本研究以体内排放的奶山羊成熟卵为供胞质的受体细胞,以人、兔、波尔山羊等的异种或亚种体细胞的原代核移植（Primary Somatic Cell Nuclear Transfer,PSCNT）重构早胚（8-16细胞期）的卵裂球作供核体,观察经亚种或异种卵胞质体短期“修饰”的核再移植产生的继代（Secondary SCNT,SSCNT）重构胚的着床前发育潜能。结果：人、兔、波尔山羊的继代桑椹／囊胚发育率均显著地高于其PSCNT胚胎（人,14．81％VS．7．79％;兔,23．53％VS．12．50％;波尔羊,55．35％VS．24．53％）;这些早胚的各阶段发育时程仍遵循供核体动物正常受精卵的发育时程。结果启示：奶山羊成熟卵胞质对异种体细胞核亦具一定的去分化能力,能支持重构胚发育到囊胚;异种重构胚的发育特征是由供体核所决定的;继代核移植几乎能够成倍提高异种间重构胚的着床前发育率,提示核的去分化完全是在母型信息主导的调控之下完成的,而进一步发育的时序似乎是由核决定的：成倍延长在含母型信息主导调控环境中的时间能成倍提高SCNT重构胚的着床前发育率。     

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。     

人-山羊异种核移植胚胎发育的初步研究

以体外分离培养的人胚胎成纤维细胞为核供体,经血清饥饿培养后,通过显微操作技术移入山羊去核卵母细胞中,采用化学方法激活重组胚.通过体外培养观察,2-细胞胚胎发育率可达51.33%,4-细胞发育率为31.42%,但发育至桑椹胚阶段的胚胎数目大大减少,仅为9.73%.虽然目前尚未能获得异种核移植囊胚,但实验结果说明山羊成熟卵母细胞可以支持人体细胞核完成重编程,人-山羊异种体细胞核移植重组胚可在体外完成其早期发育.     

人-牛异种克隆胚构建及其线粒体来源

通过人-牛异种核移植技术获得异种克隆囊胚, 便于在不消耗人类卵母细胞的情况下从异种克隆胚中分离出人类干细胞。通过透明带下注射法将人胎儿成纤维细胞和牛耳成纤维细胞分别注入去核牛卵母细胞中构建异种和同种胚胎, 并比较两者之间的融合率、卵裂率、8-细胞发育率以及囊胚率。并对处于2-细胞、4-细胞、8-细胞、桑椹胚、囊胚阶段的异种克隆胚的线粒体DNA来源进行检测。结果表明, 异种克隆胚体外各个阶段的发育率均低于同种克隆胚, 尤其是8-细胞到囊胚阶段的发育率, 以及囊胚率都显著低于同种克隆胚(P<0.05)。异种克隆胚在2-细胞到桑椹胚阶段检测到人、牛线粒体DNA共存, 囊胚阶段只检测到牛线粒体DNA。结果表明: 牛卵母细胞可以重编程人胎儿成纤维细胞, 完成异种克隆胚植入前的胚胎发育, 异种克隆胚由于核质相互作用的不谐调, 影响其发育能力, 使其囊胚率显著低于同种克隆胚。牛线粒体DNA存在于植入前异种胚胎发育的各个阶段。异种克隆胚胎用于人类胚胎干细胞分离具有可行性。     

Oct4对鼠-猪异种核移植胚胎早期发育的影响

目的探讨Oct4转录因子能否促进鼠-猪异种核移植胚胎的早期发育。方法RT-PCR获得小鼠Oct4基因,构建pEGFP-N1-Oct4-EGFP融合质粒及pEGFP-N1-Oct4-EGFP终止质粒,pEGFP-N1质粒为阴性对照,脂质体法转染小鼠NIH3T3细胞,阳性克隆经RT-PCR,荧光显微镜验证正确后,移入去核的猪卵母细胞,观察并记录发育率。结果未转染的NIH3T3阴性对照组、转染Oct4-EGFP的小鼠NIH3T3细胞和转染pEGFP-N1组均能够支持猪异种核移植胚胎的早期发育,但转染pEGFP-N1-Oct4-EGFP实验组重构胚的卵裂率和8细胞发育率与转染pEGFP-N1组和未转染的NIH3T3组的重构胚发育率差异不显著。结论NIH3T3细胞能够支持鼠-猪异种核移植胚胎早期发育,Oct4对鼠猪异种核移植胚胎的发育并没有表现出促进作用,可能也受到NIH3T3来源的异种核移植胚胎本身发育率低以及本实验室核移植显微操作水平的限制,具体的机制尚需进一步探讨。     

动物种间核移植研究概况

近年来,世界各地研究者正在努力完善异种体细胞核移植的技术环节和基础研究,本文就国外种间核移植的研究、我国哺乳动物种间核移植的研究情况进行整理分析。     

山羊M II期卵母细胞胞质支持异种间克隆胚的着床前发育

研究去核山羊(Capra hircus)体内成熟的M II期卵母细胞与异种成年的哺乳动物(包括山羊、波尔山羊、牛、塔尔羊、熊猫)及人的成纤维细胞融合形成的体细胞核移植胚胎着床前的发育能力。结果显示这些异种体细胞核移植重构胚可以完成着床前发育, 并形成囊胚。种内体细胞核移植胚的融合率和囊胚发育率分别为78.67%(557/708)和56.29%(264/469); 亚种间或种间体细胞核移植胚的融合率和囊胚发育率分别为: 波尔山羊78.18%(541/692)、33.90%(40/118), 牛70.53%(146/207)、22.52%(25/111), 塔尔羊53.51%(61/114)、5.26%(3/570), 熊猫79.82%(1159/1452)、8.35%(75/898), 人68.76%(317/461)、5.41%(16/296)。由此结果得出以下结论: (1)山羊M II期卵母细胞胞质与供核细胞之间的亲缘性不影响两者的融合率; (2)山羊M II期卵母细胞的胞质能支持异种间体细胞核移植胚的着床前发育; (3)亲缘关系近的种间核移植胚的囊胚发育率高于亲缘关系远的种间核移植胚的。     

山羊M II期卵母细胞胞质支持异种间克隆胚的着床前发育

研究去核山羊(Capra hircus)体内成熟的M II期卵母细胞与异种成年的哺乳动物(包括山羊、波尔山羊、牛、塔尔羊、熊猫)及人的成纤维细胞融合形成的体细胞核移植胚胎着床前的发育能力。结果显示这些异种体细胞核移植重构胚可以完成着床前发育, 并形成囊胚。种内体细胞核移植胚的融合率和囊胚发育率分别为78.67%(557/708)和56.29%(264/469); 亚种间或种间体细胞核移植胚的融合率和囊胚发育率分别为: 波尔山羊78.18%(541/692)、33.90%(40/118), 牛70.53%(146/207)、22.52%(25/111), 塔尔羊53.51%(61/114)、5.26%(3/570), 熊猫79.82%(1159/1452)、8.35%(75/898), 人68.76%(317/461)、5.41%(16/296)。由此结果得出以下结论: (1)山羊M II期卵母细胞胞质与供核细胞之间的亲缘性不影响两者的融合率; (2)山羊M II期卵母细胞的胞质能支持异种间体细胞核移植胚的着床前发育; (3)亲缘关系近的种间核移植胚的囊胚发育率高于亲缘关系远的种间核移植胚的。     

未经休眠处理的体细胞用于异种核移植

自“多莉”诞生以来,在全世界掀起了一场体细胞克隆的浪潮,许多体细胞克隆动物,如小鼠、山羊、牛、猪等纷纷问世。围绕体细胞克隆的供体细胞周期问题,学术界存在两种不同的观点,一是Wilmut等认为体细胞必须经过休眠处理,使细胞停滞在G0/G1期,或者采用以G0/G1期为主的活体细胞作为供体,这是克隆成功的关键,这一方面的报道已有很多。第二是Cibelli等认为不必对细胞作     

牛卵核移植技术的应用

用去核的牛卵母细胞进行核移植,形成重构胚,胚胎移植后,可产生克隆牛。将克隆牛的肉和奶制成肉粉和奶粉饲喂大鼠,大鼠的生理功能不受影响。牛卵核移植技术为珍稀动物的保护提供了一条重要途径,对于细胞治疗的研究也具有重要意义。通过牛卵核移植技术,可构建异种克隆胚胎,用以研究核质相互作用。将牛卵核移植技术和体细胞基因修饰技术相结合,可生产转基因克隆牛。     

The Fate of Mitochondria in Ibex-hirus Reconstructed Early Embryos

Nuclear transfer can be used to maintain limited popu-lations of highly endangered species [1–3] . Especiallywhen the oocytes of these species are difficult to obtain,inter-species nuclear transfer seems more appropriate forthis goal. The main methods are as follows: somatic cellis injected directly or fused by electroporation with re-cipient enucleated oocyte. Therefore, nDNA as well asmtDNA ought to be transferred totally or partially to theoocyte. Mitochondria provides adenosine triphos…     

Experimental cloning of embryos through human-rabbit inter-species nuclear transfer

Therapeutic cloning,which is based on human somatic cell nuclear transfer,is one of our major research objectives.Though inter-species nuclear transfer has been introduced to construct human somatic cell cloned embryos,the effects of type,passage,and preparation method of donor cells on embryo development remain unclear.In our experiment,cloned embryos were reconstructed with different passage and preparation methods of ossocartilaginous cell,skin fibroblast,and cumulus cells.The cumulus cell embryos showed significantly higher development rates than the other two (P＜0.05).The development rate of embryos reconstructed with skin fibroblasts of different passage number and somatic cells of different chilling durations showed no significant difference.Also,fluorescence in situ hybridization (FISH)was conducted to detect nuclear derivation of the embryos.The result showed that the nuclei of the inter-species cloned embryo cells came from human.We conclude that (1)cloned embryos can be constructed through human-rabbit interspecies nuclear transfer;(2)different kinds of somatic cells result in different efficiency of nuclear transfer,while in vitro passage of the donor does not influence embryo development;(3)refrigeration is a convenient and efficient donor cell preparation method.Finally,it is feasible to detect DNA gcnotype through FISH.     

哺乳动物核移植中供核与受体卵胞质细胞周期的相互关系

就供核与受体卵胞质细胞周期的相互关系问题进行了综述.核移植技术不管是在基础理论,还是在应用研究中都具有广泛的应用价值,但核移植的效率却很低,其根本原因是与核移植相关的许多基础理论问题尚不清楚,对这些问题的研究发现,维持重构卵核的正确倍性,并使其重新程序化是核移植成功的关键,不同的胞质受体及不同的供体细胞及其状态均对重构胚的发育有影响.     

Experimental cloning of embryos through human-rabbit inter-species nuclear transfer

Therapeutic cloning, which is based on human somatic cell nuclear transfer, is one of our major research objectives. Though inter-species nuclear transfer has been introduced to construct human somatic cell cloned embryos, the effects of type, passage, and preparation method of donor cells on embryo development remain unclear. In our experiment, cloned embryos were reconstructed with different passage and preparation methods of ossocartilaginous cell, skin fibroblast, and cumulus cells. The cumulus cell embryos showed significantly higher development rates than the other two (P < 0.05). The development rate of embryos reconstructed with skin fibroblasts of different passage number and somatic cells of different chilling durations showed no significant difference. Also, fluorescence in situ hybridization (FISH) was conducted to detect nuclear derivation of the embryos. The result showed that the nuclei of the inter-species cloned embryo cells came from human. We conclude that (1) cloned embryos can be constructed through human-rabbit interspecies nuclear transfer; (2) different kinds of somatic cells result in different efficiency of nuclear transfer, while in vitro passage of the donor does not influence embryo development; (3) refrigeration is a convenient and efficient donor cell preparation method. Finally, it is feasible to detect DNA genotype through FISH. Translated from Zoological Research, 2005, 26(4): 416–421 [译自: 动物学研究]     

Effects of donor oocytes and culture conditions on development of cloned mice embryos

Mice have been successfully cloned from somatic and embryonic stem (ES) cells using the "Honolulu method." In the present study, different donor oocytes and different culture conditions were compared to evaluate the developmental potential of nuclear transfer embryos reconstructed with an inbred ES cell line HM-1. Oocytes were recovered from two different F1 donors B6D2F1 (C57BL/6 x DBA/2) and B6CBAF1 (C57BL/6 x CBA). There was no effect of oocyte origin on development of cloned embryos to the morulae/blastocyst stage (B6D2F1 44.1% vs. B6CBAF1 45.0%), and the transferred embryos could develop to term. Two culture conditions were compared to show their ability to support development to the morulae/blastocyst stage of reconstructed embryos with B6D2F1 oocytes. The total cell number in the cloned blastocysts cultured in M16 with 20% oxygen was much higher than that observed in CZB with 20% oxygen. Low oxygen concentration during culture of nuclear transfer embryos in CZB medium showed no beneficial effect on pre-implantation development, no embryos developed to term after transfer to surrogate mothers. Our results demonstrated that not only B6D2F1, but B6CBAF1 oocytes, can be used for nuclear transfer. M16 medium is superior for culture of nuclear transfer embryos and low oxygen concentration with CZB medium during culture shows no benefit on development of cloned embryos.     

Understanding the X chromosome inactivation cycle in mice: A comprehensive view provided by nuclear transfer

During mouse development, imprinted X chromosome inactivation (XCI) is observed in preimplantation embryos and is inherited to the placental lineage, whereas random XCI is initiated in the embryonic proper. Xist RNA, which triggers XCI, is expressed ectopically in cloned embryos produced by somatic cell nuclear transfer (SCNT). To understand these mechanisms, we undertook a large-scale nuclear transfer study using different donor cells throughout the life cycle. The Xist expression patterns in the reconstructed embryos suggested that the nature of imprinted XCI is the maternal Xist-repressing imprint established at the last stage of oogenesis. Contrary to the prevailing model, this maternal imprint is erased in both the embryonic and extraembryonic lineages. The lack of the Xist-repressing imprint in the postimplantation somatic cells clearly explains how the SCNT embryos undergo ectopic Xist expression. Our data provide a comprehensive view of the XCI cycle in mice, which is essential information for future investigations of XCI mechanisms.