猪卵母细胞体外成熟的发育潜力及核移植重构胚构建方法的研究

为了提高猪体细胞核移植重构胚发育潜力,本研究对体外成熟28 h、32 h、36 h、40 h、44 h、48 h、52 h和56 h的猪卵母细胞分别进行去核构建重构胚.研究结果表明,成熟44 h的卵母细胞核移植后有较高的融合率(58.99%)、卵裂率(67.52%)和囊胚率(22.78%),而成熟48 h的卵母细胞则分别为56.51%、65.73%和15.96%;且卵龄为44 h的卵母细胞核移植后分裂率与囊胚率显著高于卵龄为40 h、36 h、32 h、28 h的卵母细胞的分裂率与囊胚率(P＜0.05).卵龄为48 h的卵母细胞融合率高于卵龄为52 h卵母细胞的融合率(P＜0.05).同时我们还探讨了不同去核方法(盲吸法、Hochest33342染色法和Spindle-view system)对猪体细胞核移植重构胚发育能力的影响.研究结果发现,盲吸法、Hoechest33342染色法和Spindle-view system法的去核率分别达到76.33%,100.00%和98.40%.Hoechest染色法去核率显著高于盲吸法的去核率(P＞0.05),而与Spindle-view法去核率没有差异(P＞0.05).三种方法在融合率和囊胚率方面差异不显著(P＞0.05),但Hoechest染色法的分裂率较低,差异显著(P＜0.05).进一步的研究表明,细胞质内注射进行核移植构建重构胚的分裂率和囊胚率分别为68.13%和6.44%;透明带下注射法则为60.37%和8.08%,两者差异不显著(P＜0.05);两者均可运用于猪体细胞的核移植,这为建立有效的猪体细胞核移植体系提供了参考.     

用小鼠血液淋巴细胞构建核移植重构胚的研究

本实验用小鼠血液淋巴细胞为核供体进行了核移植研究。用淋巴细胞分离液(比重1．088)分离出小鼠血液中的淋巴细胞,直接用作核移植供体细胞,采用胞质内注射法成功构建的重构胚经常规培养2h后,SrCl2激活处理6h,然后添加mM16培养液和小鼠输卵管上皮细胞饲养层共培养。把发育至早期囊胚阶段的重构胚转移至小鼠胎儿成纤维细胞饲养层上,添加ES细胞培养液继续培养。对孵化出的内细胞团进行消化,然后接种培养。结果显示,小鼠血液淋巴细胞可以支持体细胞核移植重构胚的发育,核移植重构胚2-细胞率41．03％(128／312),桑葚胚和囊胚发育率分别为9．29％(29／312),1．92％(6／312)。重构囊胚在小鼠胎儿成纤维细胞饲养层上分离出2个内细胞团,分离率为0．64％(2／312)。实验证实利用小鼠血液淋巴细胞进行体细胞核移植是可行的,可用于深入研究。     

体细胞的组织来源及培养代数对猪核移植重构胚发育的影响

本研究系统探讨了体细胞的组织来源及培养代数对猪核移植重构胚发育的影响。体外成熟培养40-44h的猪卵母细胞去核后,将经血清饥饿(0．5?s)培养2-9d、0．1mg/L Aphidicolin (APD)培养 0．5?S培养2-9d或一般培养法(10?S)培养的卵丘细胞、颗粒细胞、输卵管上皮细胞和耳皮成纤维细胞,直接注射到去核的卵母细胞质中,或注射到卵周隙中。再经电融合(100V/mm,30μs,电脉冲1次)构建重构胚。重构胚以钙离子载体A23817或电脉冲结合6- DMAP激活处理,体外培养6天。耳皮成纤维细胞和颗粒细胞经0．1mg/L APD 0．5?S培养处理后的重组胚卵裂率,均高于血清饥饿和一般培养处理的同种供体细胞(P＜0．01)。卵丘细胞、颗粒细胞经0．1mg/L APD 0．5?S处理后进行核移植的分裂率和发育率均高于输卵管上皮细胞和耳皮成纤维细胞(P＜0．05)。以猪颗粒细胞为核供体时,电融合法的重构胚分裂率显著高于胞质内注入法(P＜0．05),但囊胚发育率无显著差异(P＞0．05)。培养3代和6代的猪颗粒细胞以及培养6代和10代的耳皮成纤维细胞,其具有正常二倍染色体的细胞比例均无显著差异(P＞0．05);以这2种细胞不同培养代数做供体进行核移植时,各代之间核移胚的体外分裂率、囊胚发育率无显著差异(P＞0．05)。这些结果表明：(1)猪耳皮成纤维细胞和颗粒细胞经培养传代所建立起来的细胞系相对比较稳定;(2)0．1mg/L APD预培养处理供体细胞能提高猪体细胞核移植的效果,血清饥饿培养则无明显效果;(3)猪颗粒细胞和耳皮成纤维细胞等均可做供核细胞．核移植后都能得到体细胞克隆的囊胚,但前者的效果略优于后者,且其核移植效果不受供核细胞培养代数的影响;(4)电融合核移植胚胎的发育率高于胞质内直接注入法,但两者的总体效率相近。     

家兔供体细胞的发育周期与重构胚发育的关系

采用血清饥饿法处理体外培养的兔子胎儿成纤维细胞,并将其作为供体细胞移入去核卵母细胞内构建重构胚胎。检查供体细胞的细胞周期对重构胚的融合率、分裂率和着床率的影响。实验结果表明：培养基中血清含量在0．5％的情况下,G0／G1期的细胞比例由正常培养条件下(培乔液中含有10％FCS)的73．2％明显地增加到86％以上。饥饿1～3天的细胞作为供体细胞构建重构胚时,可明显提高重构胚的融合率,但是不同的饥饿时间其融合率并无显著的差异。饥饿处理可明显增加重构胚的分裂率,以饥饿处理3天为最佳。     

未经休眠处理的体细胞用于异种核移植（简报）

It is the point at issue in intraspecies nuclear transfer whether quiescence is necessary for development of nuclear transfer reconstructed embryos. In the interspecies nuclear transfer, some reports have proved that quiescent cell is able to support preimplantation development of the interspecies reconstructed embryos. Are non-quiescent cells able to support preimplantation development of the interspecies reconstructed embryos? We used non-quiescent somatic cells from C57BL/6 mice and giant pandas as donors to transfer into enucleated rabbit oocytes. After electrofusion (the electrofusion rates were 62.2% and 71.6%, respectively) and electrical activation, 5.1% of those mouse-rabbit reconstructed embryos developed to blastocyst in vitro, and 4.2% of panda-rabbit reconstructed embryos developed to blastocyst after transferring into ligated rabbit oviduct. These results indicate that non-quiescent cell from C57BL/6 mouse and giant panda could be dedifferentiated in enucleated rabbit oocytes and support early embryo development.     

山羊（Capra hircus）重构胚与再重构胚早期发育的研究

选择山羊９细胞期到桑椹胚期的正常胚胎或重构胚的卵裂球,或选择胚泡期胚胎的内细胞团细胞作为供核,并以ＬＲＨ注射后２６－２８ｈ的去核成熟卵球作为受体,制备重构胚或再重构胚,琼脂糖包埋,植入山羊输卵管内,体内培养４－６天发育结果表明：不同发育时期（８细胞期至胚泡期）的正常胚胎细胞核或重构胚细胞核中,至少有部分细胞核均保留着发育的全能性;这些细胞核在母系基因表达产物的调控下,实现＝重新编程,启动并完成正常     

枣合子胚和体细胞胚发育过程的观察与比较

本实验对临猗梨枣、壶瓶枣、晋矮1号等13个品种的枣胚的发育过程进行了观察,并诱导晋矮1号成熟胚的愈伤组织通过体细胞胚发生途径形成再生植株。结果表明:体细胞胚产生于愈伤组织的表层细胞或内部细胞。在鱼雷胚期已有导管的分化,子叶期的维管组织呈“Y”形。枣合子胚及体细胞胚的发育均经历了原胚、球形胚、心形胚、鱼雷胚和子叶胚五个时期。大多数品种的枣胚从球形胚期或心形胚期即开始败育,只有极少数品种可发育到成熟胚,而且合子胚形成的能力、胚败育时发育的程度等均存在着大的品种间差异,同一品种甚至同一子房内胚的发育进程也不同步。     

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.     

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     

不同方法处理供核细胞对细胞周期和重构胚发育的影响

利用流式细胞仪和细胞染色体核型分析技术,比较奶牛的转基因体细胞和正常细胞经血清饥饿、抑制培养周期同步化处理后的G0/G1期细胞比例;并将同步化处理的核供体细胞进行核移植,然后统计囊胚发育率.结果表明,血清饥饿和抑制培养均能获得较高比例的G0/G1期细胞,两组间差异不显著(P>0.05),但均显著高于未处理对照组(P<0.05);血清饥饿组的囊胚率显著高于抑制培养组和非处理对照组(P<0.05);但细胞同步化处理6 d后细胞染色体核型异常率增加.因此,要获得正常核型的G0/G1核移植供体细胞和较高的囊胚率,同步化处理时间以不超过4 d为宜.     

供核细胞对体细胞核移植效率的影响

利用体细胞核移植技术克隆动物、生产转基因家畜具有极大的应用潜力。然而,核移植效率低下、克隆后代形态异常等问题仍然制约着体细胞核移植技术的产业化进展。影响体细胞核移植效率的因素很多,该文着重从供核细胞的类型、细胞体外培养、细胞凋亡及转基因操作等方面阐述其对体细胞核移植效率的影响。     

牛体细胞克隆胚胎类ES细胞集落的筛选及其核移植

对第7d的牛体细胞克隆囊胚进行体外增殖培养,分离筛选类ES细胞,并对其进行了传代培养,接种在饲养层上的体细胞克隆囊胚细胞,在传代的24h内增殖形成小集落,2～3d有雀巢状的集落出现,筛选形态相同的细胞集落进行传代培养,4～5代后,皿底出现多个大小不等的多细胞单层集落,将传4～5代的细胞集落接种到无饲养层的4孔培养皿中培养,24h出现多细胞单层集落,4～7d长满皿底,并形成上皮样细胞,呈网状,将其作为核供体细胞进行核移植实验。结果有80%（40/50）核-质融合的移核重构胚发生卵裂,5%（2/40）发育至桑椹胚期,2.5%（1/40）发育至囊胚期,92.5%（37/40）停止在2～4细胞期,结果表明：采用牛体细胞克隆胚胎的类ES细胞进行核移植,具发育形成早期胚胎的潜能。     

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…     

体细胞核移植胚胎核重编程的研究进展

尽管在多种哺乳动物种系中成功制备了体细胞克隆后代,但当前的克隆技术仍有许多亟待解决的问题。体细胞核移植胚胎大多存在许多发育异常,造成了妊娠早期高流产率和出生后高死亡率。有研究认为,克隆胚胎发育障碍的一个重要的原因是供体细胞的遗传重编程不完全。哺乳动物种系中,DNA甲基化是胚胎发育期转录调节的必需步骤,除了单拷贝基因序列外,在基因组很多的区域都可以观测到克隆胚胎的异常甲基化。此外,克隆胚胎的基因印迹也存在异常。     

Generation of cloned calves from different types of somatic cells

Six types of bovine somatic cell lines, including a granulosa cell line of Chinese red-breed yellow cattle (YGR), a granulosa cell line of Holstein cow (HGR), two skin fibroblast cell lines of two adult Holstein cows respectively (AFB1 and AFB2), a skin fibroblast cell line (FFB) and an oviduct epithelial cell line (FOV) of a Holstein fetus, were established. Somatic cell nuclear transfer (SCNT) was carried out using these cells as nuclei donor, and a total of 12 healthy calves were cloned. The effects of different types of donor cells on developmental potential of bovine SCNT embryos were investigated, (i) There was no significant difference in development rates to the blastocyst stage for SCNT embryos from YGR and HGR (33.2% and 35.1%, respectively). Pregnancy rates of them were 33.3% and 30.2%, respectively; and birth rates were 16.7% and 11.6%, respectively, (ii) Development rates to the blastocyst stage for SCNT embryos from diffetent individuals (AFB1 and AFB2) differed significantly (27.9% and 39.4%, respectively, P < 0.05). Pregnancy rates of them were 36.2% and 36.4%, respectively; and birth rates were 14.9 % and 27.3%, respectively, (iii) There was significant difference in development rates to the blastocyst stage for SCNT embryos from FFB and FOV of the same fetus (37.9% and 41.5%, respectively,P < 0.05). Pregnancy rates of them were 45.7% and 24.1%, respectively; and birth rates were 22.9 % and 10.3%, respectively. Finally, developmental potential of bovine SCNT embryos from all four types of somatic cells from Holstein cows (HGR, AFB, FFB and FOV) were compared. Forin vitro development stage, development rates to the blastocyst stage for SCNT embryos from HGR, AFB, FFB and FOV were 35.1%^A, 29.4%^B, 37.9%^A and 41.5%^C, respectively (P ^ABC < 0.05); forin vivo development stage, pregnancy rates of them were 30.2%, 36.2%, 45.7% and 24.1%, respectively; and birth rates of them were 11.6%, 17.2%, 22.9% and 10.3% respetively.     

哺乳动物体细胞核移植中供体细胞的研究进展

在哺乳动物体细胞核移植中,供体细胞是影响其效率的主要因素之一。供体细胞的类型、细胞周期、细胞的培养代数、冷藏与冷冻处理,以及供体动物的性别、年龄等都可能影响核移植胚胎的发育。根据现有资料,简要综述了在哺乳动物体细胞核移植中有关供体细胞的研究进展。     

Age-Related Effect of Cells as Donors of Nuclei: On the Efficiency of the Development of Cloned Rabbit Embryos

We studied the capacity of the nuclei of rabbit fibroblasts taken from various developmental stages for reprogramming in the cytoplasm of mature aging enucleated oocytes and the development of the cloned embryos to the preimplantation stages. A negative correlation was found between the age of an animal donor of fibroblasts and the efficiency of the development of cloned embryos (r _{morula-blastocyst}= –0.826, r _blastocyst= –0.7139). A reliably decreased capacity for reprogramming of the nuclei of donor fibroblasts was shown upon the transition from prenatal development to postnatal development, as well as a trend to a decreased capacity of nuclei for reprogramming during aging. The aging of cells in the culture, at least until the tenth passage, did not affect the capacity of the nuclei of fetal fibroblasts for reprogramming and the development of cloned embryos.     

鸡蛋开窗法导入供体胚盘细胞对家鸡胚胎发育的影响研究

通过4批孵化实验,研究鸡蛋开窗法注射供体胚盘细胞对受体鸡胚发育及孵化率的影响。GLM方差分析表明,开窗处理极显著降低整个孵化期（21d)的活胚比例（P＜0．01）,至21日龄出壳时,处理组的孵化率为.8%-6.0%。导入供体胚盘细胞对受体鸡胚的发育仅为阶段性影响,表现在显著性孵化第8日龄左右的活胚比例（P＜0.05)。而对后期发育的影响不显著（P＞0．05）。因经,鸡蛋开窗处理是降低受体胚胎成活率和孵化率的主要原因,如何降低开窗处理对受体胚胎的应激和不利影响则是今后研究的一个课题。     

Effects of donor cells on in vitro development of cloned bovine embryos

The donor cells from different individuals and with different foreign genes introduced were investigated to determine their effects on the efficiency of somatic cell nuclear transfer (SCNT). The bovine ear fibroblast from different individuals was isolated, cultured, and then transfected with foreign genes to establish the stable cell lines, which were used as donor cells for nuclear transfer. The ooeytes were obtained through ovum pick up operation. After in vitro maturation, the M II phase oocytes were selected as receptors for nuclear transfer.The reconstructed embryos were cultured in vitro and observed at 2 h, 48 h, and 7 days after transfer to assess the rate of fusion using cleaved and blastoeyst as the parameters of SCNT efficiency. The donor cells from different individuals (04036, 06081, 06088, and 06129)had no obvious effect on the fusion and cleaved rate, whereas there was significant difference in the blastocyst rate (P<0.05), and the rate was 62.3%, 37.0%, 35.1%, and 15.6%, respectively. There was no significant difference among the rate of fusion, cleaved and blastocyst in donor cells with different foreign genes (P>0.05). It was concluded that the genetic background of the donor cells could affect the effi-ciency of SCNT, while the introduction of foreign genes into the donor cells had no obvious effect on the efficiency. This study provides useful information for the SCNT and would benefit in promoting the efficiency.