不同受体胞质对延边黄牛体细胞核移植效率的影响

为确定延边黄牛体细胞核移植过程中卵母细胞的最佳去核时期,本试验选取体外成熟22 h的中期(MⅡ期)卵母细胞与体外成熟28 h的末期(TⅡ期)卵母细胞进行化学辅助去核操作,并将去核的卵母细胞作为受体构建重构胚,后期观察发育情况.结果显示,M期卵母细胞的显核率与TⅡ期卵母细胞的显核率无显著差异;但是TⅡ期卵母细胞所得重构胚的卵裂率及囊胚率均显著高于MⅡ期卵母细胞所得重构胚的卵裂率及囊胚率.综上所述,卵母细胞体外成熟28 h后的TⅡ期可作为延边黄牛核移植去核操作的理想时期,在TⅡ期卵母细胞中选择化学辅助去核法去核能够提高延边黄牛体细胞克隆的效率.     

鼠、兔核质杂交及早期胚胎发育的研究

以2~8细胞期鼠胚细胞为供体核,选用注射hCG后15h、17h的兔卵母细胞为受体胞质,施加1.6kv/cm、160、两次脉冲的电场条件,间隔20min,融合液为含Ca、Mg的0.3mol/L甘露醇,适于鼠、兔种间EOBC的融合激活。小鼠2-细胞、4-细胞期胚细胞与兔去核卵母细胞融合率高于8-细胞期胚细胞,分别为83%、80%及62.5%,桑囊胚发育率差异不显著。体细胞共同培养系统和Taurine能显著提高鼠、兔核质杂交胚的体外发育能力。     

化学辅助去核法对延边黄牛卵母细胞去核率及其重构胚发育率的影响

为提高延边黄牛体细胞克隆的效率,采用化学辅助去核法--化学诱导剂脱羰秋水仙碱(Demeeoleine,简称Deme)处理体外成熟的MⅡ期延边黄牛卵母细胞,经显微操作构建重构胚后观察发育情况.试验研究了,Deme的处理浓度、作用时间对延边黄牛卵母细胞去核效果及其重构胚发育的影响.结果显示:体外成熟的卵母细胞在0.2μg/mL、0.3 μg/mL、0.4 μg/mL的Deme溶液中处理45 min,显核率与去核率无显著差异,去核率达100%,但是0.2 μg/mL处理组的囊胚发育率显著高于其他处理组;45 min处理组的显核率显著高于30 min、60 min处理组,45 min处理组的囊胚率高于其他处理组;化学辅助去核法的囊胚率显著高于盲吸法.化学辅助去核能够准确定位去除细胞核,并提高了延边黄牛体细胞克隆的去核率及重构胚的体外发育率.     

家兔胚胎细胞核移植的研究

兔超排后收集卵母细胞和１６细胞胚，以Ｍｃ－Ｇｒａｔｈ－Ｓｏｌｔｅｒ和Ｗｉｌｌａｄｓｅｎ两种去（注）核方法，将１６细胞胚细胞核经电融合植入去核成熟卵母细胞内，经体外培养或中间受体培养，使移核胚发育．结果表明：（１）兔胚核移植中上述两种去（注）法的成功率无差异；（２）ｈｃＧ注射后１３～１５ｈ，６７．８％的卵母细胞保留有第一极体；（３）强度为０．６３ｋＶ／ｃｍ，持续１６０μｓ的一次电脉冲可使７０．８％的注核胚融合，同样的电脉冲刺激卵母细胞的活化率为６１．１％；（４）兔移核胚在中间受体内或体外均可发育，在中间受体内有２３．０％可以发育到桑椹胚或囊胚．     

提高山羊卵丘细胞核移植效果的研究

 【目的】提高山羊卵丘细胞核移植效果。【方法】采用秋水仙胺提高山羊卵母细胞去核率,5-氮-2'-脱氧核苷（5-aza-dC）和曲古菌素A（TSA）影响山羊卵丘细胞核移植胚胎。【结果】0.5 μg?ml-1秋水仙胺处理山羊卵母细胞效果最好,胞质突起率达91.7%（P＜0.05）;通过比较盲吸去核法与化学辅助去核法的去核率及构建卵丘细胞核移植胚胎的体外发育能力发现,化学辅助去核法的去核率达100%,所构建的重构胚体外发育能力较高,桑椹胚率和囊胚率分别达25.2%和13.1%,显著高于盲吸去核法（P＜0.05）。采用0.01 μmol?L-1的5-aza-dC处 理山羊卵丘细胞,核移植胚胎桑椹胚率和囊胚率分别达30.4%和17.0%,显著高于其它各组（P＜0.05）;采用 400 nmol?L-1TSA处理山羊卵丘细胞,核移植胚胎桑椹胚率、囊胚率分别达31.5%和15.2%。【结论】化学辅助去核法的去核率显著高于盲吸去核法,采用0.01 μmol?L-1的5-aza-dC或400 nmol?L-1 TSA处理山羊卵丘细胞,效果最佳。     

山羊卵核移植的研究

 选用陕北黑山羊的4-32细胞胚和萨能奶山羊的次级卵母细胞分别作核供体和胞质供体。在显微操作仪的控制下，将分离后的单个卵裂球注入去核次级卵母细胞的卵周隙。采用电融合法诱导卵裂球和去核次级卵母细胞融合。通过体外培养和移植，检查卵核移植的效果。本研究证明：⑴在4-32细胞期，核供体胚的发育阶段对山羊卵核移植胚的体外发育无显著影响；⑵由山羊4-32细胞期胚胎的卵裂球和去核次级卵母细胞构成的卵核移植胚在体内仍可发育为正常个体；⑶用注射LH后25-35小时的次级卵母细胞作胞质供体均可获得较高的卵核移植胚体外发育率；⑷用900伏/厘米的电场强度进行2个80微秒的电脉冲处理，诱导卵裂球和去核次级卵母细胞融合，可获得更好的电融合效果。根据山羊胚胎性RNA在2细胞期出现的事实和本研究的上述结果，作者认为，次级卵母细胞的胞质对移入的晚期卵裂胚细胞核可能有去分化作用。     

化学去核卵母细胞为受体的小鼠体细胞核移植

 【目的】本研究旨在探索一种崭新的、化学试剂诱导去核卵母细胞为核受体的、无透明带的、手工体细胞核移植方法。【方法】将第一次减数分裂期小鼠卵母细胞进行诱导去核并去除透明带，去核卵胞质与胎儿成纤维细胞粘合、电融合和SrCl2激活后，体外培养重构胚。【结果】重构胚融合率和激活率分别为84.8％和93.6％；胚胎2-细胞发育率为24.7％，4-细胞率为6.74％；2-细胞期克隆胚移植假孕受体后，没有获得怀孕受体；分别以“血清饥饿”胎儿成纤维细胞、新鲜细胞和冷冻保存细胞为供体作核移植，结果表明，冷冻保存细胞的融合率（69.3％）与其余两组（80.6％和84.8％）呈显著差异（P＜0.05）；激活率、2-细胞和4-细胞发育率，则3组间差异不显著（P＞0.05）。【结论】本文将小鼠卵母细胞的化学去核与无透明带技术相结合，获得的克隆胚目前已发育到4－细胞期；另外，供体细胞的3种准备方式均不影响胚胎发育率。该方法属手工克隆，它的成功将会大大简化核移植程序，提高核移植总效率。     

家兔胚胎细胞核移植的研究

 以兔8-16细胞的新鲜胚胎或冷冻-解冻胚胎的卵裂球作为细胞核供体，成熟的去核卵母细胞作为细胞核受体。经显微操作，将分离的单个卵裂球移入去核卵母细胞透明带内，制成322枚组合卵。对组合卵施加一个1200伏/cm（V/cm）和80微秒（μs）的直流电脉冲，使卵裂球的细胞核导入去核卵母细胞之中。由新鲜供体胚胎或冷冻-解冻供体胚胎与去核卵母细胞构成的组合卵的融合率分别为87.2%(251/288)和73.5%(25/34)。将部分已融合的细胞核移植(Nuclear Transplantation，NT)卵置于含10%胎犊血清(FCS)的TCM199培养液中，在充以5%CO#-2+空气的培养箱内，于39℃下培养20小时，新鲜供体胚胎和冷冻-解冻供体胚胎的NT卵发育至2-4细胞的分裂率分别为58.0%(47/81)和53.8%(7/13)。将53枚NT卵移入同步发情的5只受体母兔的输卵管内，结果一只母兔于妊娠第33天经剖腹手术产出1只体重为70克雌性核移植仔兔。实验结果表明，兔8-16细胞阶段的新鲜胚胎和冷冻-解冻胚胎均可在细胞核移植中用作细胞核供体。     

家兔胚胎细胞核移植的研究

研究改进了兔胚胎细胞核移植的技术体系,并对核移植胚胎的体外培养以及克隆胚胎的体内发育进行了试验。结果表明：卵母细胞去核率为８８．９％（８／９）,重组胚融合率为７６．７％（１７６／２２９）,将融合的重组胚与兔胎儿成纤维细胞共同培养,其２￣４细胞发育率、８￣１６细胞发育率以及桑椹胚和囊胚的发育率分别为６５．７％（２３／３５）、２８．６％（１０／３５）、２２．９％（８／３５）,显著高于“ＴＣＭ－１９９＋１０％ＦＣＳ”系统培养的４３．３％（１３／３０）、２６．７％（８／３０）、１６．７％（５／３０）。１６及３２细胞期卵裂球的重组胚可发育到产仔,将１１１枚重组胚移植给９只同期处理的受体母兔,２只妊娠,其中１只产出２只足月克隆仔兔,１只在产前１周（妊娠２２ｄ）流产。     

影响兔胚胎细胞核移植效率的因素

 对兔胚胎细胞核移植（NT）的有关影响因素进行了系统研究。结果发现电场强度100 V·mm-1，脉冲时间15μs，电脉冲3～4次的融合率显著高于其它组（P<0.05）；融合前激活卵母细胞，分裂率和囊胚率显著高于融合后激活（P <0.05）；当用8～16-细胞胚胎的卵裂球作供核时，卵裂率和囊胚率显著高于致密桑椹胚卵裂球为供核组；当重组胚在含3%发情牛血清（OCS）的TCM199中培养48 h后，转入含10% FCS的TCM199中继续培养，卵裂率和囊胚率显著高于一直在3% OCS或10% 胎犊血清（FCS）中培养的重组胚（P < 0.05）。将22枚2～4-细胞期重组胚移入同期发情的受体母兔输卵管内，34 d后产下NT仔兔1只。结果表明，电融合参数和供体胚胎的发育阶段以及受体卵母细胞的状态对NT效果有显著影响，在NT胚胎的不同发育阶段采用不同的血清种类和浓度可提高其胚胎发育能力。     

猪胚胎细胞核移植

选用杜洛克２～１６细胞期胚胎卵裂球作核供体，湖北白猪卵母细胞作核受体，通过显微操作和电融合法构成重组胚。体外培养时，以融合前２ｈ，１ｈ激活及融合前不激活的卵母细胞做核受体的重组胚，发育率分别为６４．６％（３１／４８），５５．３％（２６／４７）和３４．５％（１９／５５）。６１枚重组胚移入同步发情的５头受体母猪输卵管，１头于妊娠１１７ｄ产下５头核移植仔猪。结果表明，激活卵母细胞作核受体优于未激活卵母细胞。成熟卵母细胞的胞质对于移入的核具有重排能力     

In Vitro Developmental Potential of Cloned Embryos Derived from Bovine Somatic Cells and Rabbits Oocyte

180 reconstituted embryos were produced by nuclear transplantation using bovine ear fibroblasts at G0 or non-G0 stage as donor nuclei and oocytes collected from superovulated multiparous or young rabbits as recipients. After cultivation in two kinds of medium M199+ 10%FBS or RD+ 10%FBS, 112 of them developed to 2-cell stage (62.2%) and 26 to morula stage (14.4%) and 20 of them eventually developed to blastocyst stage (11. 1% ). There is no significant difference for the cleavage rates in two groups of reconstituted embryos derived from G0-stage and non-G0 stage donor cells respectively. However, G0-stage donor cells could result in higher rate of 8-cell - 16-cell stage embryos significantly (P＜0.05), as well as higher rate of blastocysts (P＜0.01). It seems that using two different culture systems had no significant effects on the cleavage rate, morula rate or blastocyst rate (P＞0.05).     

不同培养条件对牛核移植胚胎体外发育的影响

[目的]进一步优化牛核移植胚胎的体外培养体系。[方法]在培养液中添加谷胱甘肽和胚胎培养的0~3 d采用5%O2浓度,在不同培养条件下对牛核移植重构胚胎进行体外培养,测定卵裂率8、细胞发育率、桑椹胚率以及囊胚发育率。[结果]结果表明,试验组B的8细胞发育率(38.3%)、桑椹胚率(17.0%)和囊胚率(8.5%)与对照组A(17.9%、1.3%、1.3%)差异显著(P<0.05)。试验组B和试验组C的桑椹胚率(17.0%、11.8%)、囊胚率(8.5%、7.8%)与对照组A(1.3%、1.3%)均差异显著(P<0.05)。[结论]在培养液中添加谷胱甘肽和胚胎培养的0~3d采用低O2浓度(5%O2),能够提高牛核移植胚胎的体外发育率。     

Factors Affecting the Efficiency of Embryonic Cell Nuclear Transfer in Rabbits

Factors affecting the efficiency of nuclear transfer （NT） in rabbits were examined in the present study. When 100 V mm of pulse strength and 15 us of pulse duration were employed, 3 and 4 electronic pulses resulted in significantly more cytoplasts fused with donor cells compared with 2 electronic pulses （P〈 0.05）, but no significant difference was found in the cleavage rate of reconstructed embryos among the three groups （P〉0.05）. When the duration and number of electronic pulse were fixed at 15 ps and 3 times, increase of pulse intensity from 100 V mm 1 to 150 V mm^-1 and 200 V mm^-1 resulted in a significantly decrease in the cleavage rate of reconstructed embryos （P〈 0.05）, although the fusion rate did not significantly differ among the three groups （P〉 0.05）. Significantly more reconstructed embryos cleaved and developed to blastocysts when they were derived from donor embryos at the 8-16-cell stage, in comparison with the reconstructed embryos derived from donor embryos at the compact morula stage （P 〈 0.05）, although the fusion rate was similar （P 〉 0.05）. Activation of cytoplasts prior to fusion increased the cleavage rate （P〈 0.05） and blastocyst development （P〈 0.05） of reconstructed embryos, but decreased the fusion rate （P 〈 0.05） compared with cytoplasts activated post fusion. More reconstructed embryos developed to blastocysts when they were cultured in TCM ＋ 3% OCS at the first 48 h and then cultured in TCM199＋ 10% FCS, in comparison with the reconstructed embryos cultured in either TCM199＋ 10% FCS or TCM199＋3% OCS （P 〈 0.05）. When 22 NT embryos were transferred into the oviducts of one recipient rabbit, one recipient rabbit delivered a female rabbit at 34 days of gestation. In conclusion, either electrofusion parameter or developmental stage of donor embryos have a significant effect on the efficiency of NT, NT embryos require different concentration of serum at their different development stages.     

Patient-specific embryonic stem cells derived from human SCNT blastocysts

Patient-specific, immune-matched human embryonic stem cells (hESCs) are anticipated to be of great biomedical importance for studies of disease and development and to advance clinical deliberations regarding stem cell transplantation. Eleven hESC lines were established by somatic cell nuclear transfer (SCNT) of skin cells from patients with disease or injury into donated oocytes. These lines, nuclear transfer (NT)-hESCs, grown on human feeders from the same NT donor or from genetically unrelated individuals, were established at high rates, regardless of NT donor sex or age. NT-hESCs were pluripotent, chromosomally normal, and matched the NT patient's DNA. The major histocompatibility complex identity of each NT-hESC when compared to the patient's own showed immunological compatibility, which is important for eventual transplantation. With the generation of these NT-hESCs, evaluations of genetic and epigenetic stability can be made. Additional work remains to be done regarding the development of reliable directed differentiation and the elimination of remaining animal components. Before clinical use of these cells can occur, preclinical evidence is required to prove that transplantation of differentiated NT-hESCs can be safe, effective, and tolerated.     

In vivo and in vitro development of Tibetan antelope (Pantholops hodgsonii) interspecific cloned embryos

The Tibetan antelope is endemic to the Tibetan Plateau, China, and is now considered an endangered species. As a possible rescue strategy, the development of embryos constructed by interspecies somatic cell nuclear transfer (iSCNT) was examined. Tibetan antelope fibroblast cells were transferred into enucleated bovine, ovine and caprine oocytes. These cloned embryos were then cultured in vitro or in the oviducts of intermediate animals. Less than 0.5% of the reconstructed antelope-bovine embryos cultured in vitro developed to the blastocyst stage. However, when the cloned antelope-bovine embryos were transferred to caprine oviducts, about 1.6% of the embryos developed to the blastocyst stage. In contrast, only 0.7% of the antelope-ovine embryos developed to the morula stage and none developed to blastocysts in ovine oviducts. The treatment of donor cells and bovine oocytes with trichostatin A did not improve the embryo development even when cultured in the oviducts of ovine and caprine. When the antelope-bovine embryos, constructed from oocytes treated with roscovitine or trichostatin A, were cultured in rabbit oviducts 2.3% and 14.3% developed to blastocysts, respectively. It is concluded that although some success was achieved with the protocols used, interspecies cloning of Tibetan antelope presents difficulties still to be overcome. The mechanisms resulting in the low embryo development need investigation and progress might require a deeper understanding of cellular reprogramming.     

不同培养液对牛体细胞核移植胚胎发育的影响

旨在为牛体细胞克隆胚胎体外培养体系的建立奠定基础。利用体细胞核移植技术生产牛重构胚,将重构胚随机分成2组,分别用mSOF和G1.5/G2.5培养,统计2种培养液对牛核移植克隆胚胎发育的影响,用TUNEL荧光检测系统检测2个培养组胚胎细胞凋亡情况,同时用Real time RT-PCR检测热应激蛋白基因Hsp70和凋亡相关基因Bax在2个培养组的相对表达水平。两组的卵裂率分别为85.6%和87.0%、囊胚率分别为22.9%和27.1%,差异都不显著(P>0.05),但G1.5/G2.5组的8-细胞形成率显著高于mSOF组(P<0.05);mSOF组凋亡率为12.1%±1.9%,显著高于G1.5/G2.5组(P<0.05),G1.5/G2.5组的Hsp70和Bax相对表达量显著低于mSOF组(P<0.05)。结果表明,G1.5/G2.5培养液更有利于牛体细胞核移植胚胎的体外发育。     

微滴中胚胎数量对猪孤雌胚早期体外发育的影响

[目的]优化猪早期胚胎培养体系,为单精子显微受精及体细胞核移植等研究提供依据。[方法]以猪的早期孤雌胚胎为材料,分别将人工激活后的70、50、30、15和5枚卵母细胞放入100μlNCSU-23培养液的微滴中进行培养,探讨了胚胎培养数量对猪早期孤雌胚发育的影响。[结果]100μl的培养微滴中培养30、50和70胚胎组,其分裂率分别为64.0%、65.2%和67.1%,显著高于5胚胎组,而与15胚胎组的差异不显著 在囊胚率方面,70胚胎组的最高,为3.0%,与50胚胎组的（1.7%）无显著差异,与5、15和30胚胎组的差异显著 各组的囊胚细胞数之间没有显著差异。[结论]在该研究条件下,当微滴体积为100μl时,培养50~70枚猪孤雌胚胎的效果最好,即,胚胎数∶培养滴体积=（1∶1.43~2.00）。