5-Aza-CdR对德保黑猪手工克隆重构胚胎体外发育效果的影响

为了探讨5-氮杂-2'-脱氧胞苷（5-Aza-CdR）对德保黑猪手工克隆（HMC）重构胚胎体外发育效果的影响，本研究分别从供体细胞和重构胚入手，比较了5个不同处理浓度（0、5、10、20和40 nmol/L）5-Aza-CdR处理HMC重构胚的体外发育效果，筛选最佳处理浓度；在最佳浓度下比较5个不同处理时间（0、24、48、72和96 h）对HMC重构胚的体外发育效果，筛选最佳处理时间；用4个不同浓度（0、0.25、0.5和1 μmol/L）5-Aza-CdR结合最佳浓度和最佳时间处理供体和重构胚，比较其体外发育潜能。结果显示，与空白对照组相比，5、10、20和40 nmol/L 5-Aza-CdR处理72 h对重构胚卵裂率均无显著差异（P>0.05），20 nmol/L 5-Aza-CdR处理能显著提高重构胚的囊胚率（P<0.05），10和20 nmol/L 5-Aza-CdR处理均能显著提高囊胚细胞数（P<0.05），其中以20 nmol/L 5-Aza-CdR效果最佳；与空白对照组相比，利用20 nmol/L 5-Aza-CdR处理HMC重构胚72 h能显著提高重构胚的囊胚率和囊胚细胞数（P<0.05），其余处理时间对重构胚卵裂率、囊胚率和囊胚细胞数均无显著影响（P>0.05）；在囊胚的最佳处理浓度（20 nmol/L）和最佳处理时间（72 h）下，结合供体的4个处理浓度（0、0.25、0.5和1 μmol/L），同时处理重构胚和供体，各处理组HMC重构胚的发育潜能均有提高，但效果均不显著（P>0.05），其中0.25~0.5 μmol/L 5-Aza-CdR处理效果较佳。综上表明，适宜浓度（0.25~0.5 μmol/L）的DNA甲基化酶抑制剂5-Aza-CdR处理供体细胞72 h并结合20 nmol/L 5-Aza-CdR处理重构胚72 h均能有效提高德保黑猪HMC重构胚胎的体外发育潜能，该结果可为今后研究德保黑猪HMC胚胎DNA甲基化调控机制提供参考。     

5-Aza-CdR对德保黑猪手工克隆重构胚胎体外发育效果的影响

为了探讨5-氮杂-2′-脱氧胞苷(5-Aza-CdR)对德保黑猪手工克隆(HMC)重构胚胎体外发育效果的影响,本研究分别从供体细胞和重构胚入手,比较了5个不同处理浓度(0、5、10、20和40nmol/L)5-Aza-CdR处理HMC重构胚的体外发育效果,筛选最佳处理浓度;在最佳浓度下比较5个不同处理时间(0、24、48、72和96h)对HMC重构胚的体外发育效果,筛选最佳处理时间;用4个不同浓度(0、0.25、0.5和1μmol/L)5-Aza-CdR结合最佳浓度和最佳时间处理供体和重构胚,比较其体外发育潜能。结果显示,与空白对照组相比,5、10、20和40nmol/L5-Aza-CdR处理72h对重构胚卵裂率均无显著差异(P0.05),20nmol/L 5-Aza-CdR处理能显著提高重构胚的囊胚率(P0.05),10和20nmol/L 5-Aza-CdR处理均能显著提高囊胚细胞数(P0.05),其中以20nmol/L 5-AzaCdR效果最佳;与空白对照组相比,利用20nmol/L 5-Aza-CdR处理HMC重构胚72h能显著提高重构胚的囊胚率和囊胚细胞数(P0.05),其余处理时间对重构胚卵裂率、囊胚率和囊胚细胞数均无显著影响(P0.05);在囊胚的最佳处理浓度(20nmol/L)和最佳处理时间(72h)下,结合供体的4个处理浓度(0、0.25、0.5和1μmol/L),同时处理重构胚和供体,各处理组HMC重构胚的发育潜能均有提高,但效果均不显著(P0.05),其中0.25～0.5μmol/L 5-Aza-CdR处理效果较佳。综上表明,适宜浓度(0.25～0.5μmol/L)的DNA甲基化酶抑制剂5-AzaCdR处理供体细胞72h并结合20nmol/L 5-Aza-CdR处理重构胚72h均能有效提高德保黑猪HMC重构胚胎的体外发育潜能,该结果可为今后研究德保黑猪HMC胚胎DNA甲基化调控机制提供参考。     

克隆山羊成纤维细胞IGF2-H19基因座甲基化分析

旨在研究山羊体细胞核移植对克隆后代成纤维细胞IGF2-H19基因座甲基化的影响。以奶山羊耳成纤维细胞(GFC,对照组)和克隆山羊耳成纤维细胞(CFC,试验组)为试验材料,培养至第5代时,采用细胞计数法绘制细胞生长曲线,流式细胞仪检测细胞凋亡情况,实时荧光定量PCR分析基因的表达差异,亚硫酸氢盐测序(BSP)分析差异甲基化区域的甲基化水平。结果显示,GFC和CFC组细胞的生长曲线均呈典型"S"型,但CFC组细胞的凋亡率显著高于GFC组(P0.01);CFC组细胞中Dnmt1(P0.01)、Dnmt3b(P0.01)、Tet1(P0.05)、Tet2(P0.05)、H19(P0.05)和IGF2(P0.01)基因表达水平均显著低于GFC组,而Tet3、Dnmt3a和IGF2R在2组间无显著性差异;CFC组细胞中IGF2两个差异甲基化区域(DMR1和DMR2)的甲基化水平均显著低于GFC组(74.1%vs.57.8%,P0.01;76.8%vs.40.0%,P0.01),但IGF2-H19印记基因控制区域甲基化水平显著高于GFC组(68.8%vs.84.0%,P0.01)。体细胞核移植通过影响Dnmt和Tet家族基因的表达引起IGF2-H19基因座甲基化的异常,导致基因印记紊乱,进而影响再克隆的效率。     

猕猴外周血液中多潜能调控因子Oct4、Nanog和Sox2基因的表达

采用半定量RT-PCR对采自幼体(2岁以内)、亚成体(2～5岁)和成体(5岁以上)猕猴共28份外周血样品进行分析.结果显示,幼体和亚成体猕猴所有外周血样品均转录表达Oct4、Nanog和Sox2基因,大部分成体猕猴外周血样品(8/12)表达Oct4、Nanog和Sox2基因,少数样品仅表达Oct4和Sox2(2/12)或Oct4和Nanog(1/12),1份样品不表达3个基因;同一年龄组内,Sox2表达水平均显著高于Oct4和Nanog(P≤0.05),幼体组Oct4和Nanog的表达水平没有显著性差异(P＞0.05),亚成体组和成体组Oct4的表达水平均显著高于Nanog(P≤0.05);在不同年龄组间,Oct4和Nanog表达水平没有显著性差异(P＞0.05),幼体组Sox2表达水平显著高于成体组(P≤0.05).结果表明,在生理条件下Oct4、Nanog和Sox2基因在不同年龄段猕猴外周血中均转录表达,不同基因表达水平存在一定差异,随年龄增加这些基因表达量有降低的趋势.     

母源基因Gdf9、Zar1、Mater和Dnmt1mRNA在绵羊卵母细胞和早期胚胎中的表达

为探讨母源基因在绵羊卵母细胞和胚胎发育过程中的表达模式,运用Real-time PCR技术研究4种母源基因:Gdf9(生长分化因子9)、Zar1(合子阻泄因子)、Mater(胚胎必要的母体抗原)及Dnmt1(DNA甲基化转移酶1)的mRNAs在绵羊GV期卵母细胞,24h成熟卵母细胞,2-细胞、4-细胞、8-细胞、16-细胞胚胎以及囊胚中的含量变化情况。结果表明:在GV期卵中Zar1、Mater、Gdf9以及Dnmt1m RNA相对含量最高(P<0.05);从2-细胞胚胎开始,4种基因的mRNA相对含量显著降低(P<0.05),各基因mRNA的含量在不同发育阶段的卵母细胞和胚胎中存在动态变化,这对卵子生长和胚胎发育有重要意义。     

Nanog基因的生物学功能研究进展

胚胎干细胞具有无限增殖能力和多向分化潜能决定了它在医学及生物学基础研究中具有巨大的应用潜力。探索维持胚胎干细胞特性的分子机制成为胚胎干细胞的生物学研究中的热点。研究发现与维持胚胎干细胞多能性相关的基因有Oct4、Nanog、Sox2等，其中Nanog是2003年5月末发现的一个基因，它对维持胚胎干细胞多能性起关键性作用，能够独立于L1F/Stats维持ICM和ES细胞的多能性。几年来，Nanog的生物学功能及其与Oct4、Sox2等多能性维持基因之间的相互作用关系已有较为深入的研究。作者在综述Nanog基因的表达特征和功能的基础上，重点探讨Nanog基因表达调控以及Oct4、Sox2等多能性维持基因之间的相互作用关系，并展望其应用前景。     

母源基因Gdf9、Zar1、Mater和Dnmt1 mRNA在绵羊卵母细胞和早期胚胎中的表达

为探讨母源基因在绵羊卵母细胞和胚胎发育过程中的表达模式,运用Real-time PCR技术研究4种母源基因:Gdf9(生长分化因子9)、Zar1(合子阻泄因子)、Mater(胚胎必要的母体抗原)及Dnmt1(DNA甲基化转移酶1)的mRNAs在绵羊GV期卵母细胞,24h成熟卵母细胞,2-细胞、4-细胞、8-细胞、16-细胞胚胎以及囊胚中的含量变化情况。结果表明:在GV期卵中Zar1、Mater、Gdf9以及Dnmt1m RNA相对含量最高(P0.05);从2-细胞胚胎开始,4种基因的mRNA相对含量显著降低(P0.05),各基因mRNA的含量在不同发育阶段的卵母细胞和胚胎中存在动态变化,这对卵子生长和胚胎发育有重要意义。     

细胞松弛素B对猪孤雌囊胚DNA甲基化和组蛋白乙酰化的影响

以猪电激活孤雌胚胎为研究对象,通过添加细胞松弛素B(CB)对胚胎发育过程中DNA甲基化和组蛋白乙酰化的变化进行了研究。利用荧光定量PCR检测了囊胚中DNA甲基转移酶(Dnmt1,Dnmt3a,Dnmt3b)、组蛋白乙酰转移酶(Hat1)、组蛋白去乙酰化酶(Hdac1)以及凋亡相关基因(Bax,Casp3)mRNA水平的表达,并通过免疫荧光染色检测了囊胚中5hmc/5mc,AcH3K9,H3K9me3的表达水平。结果显示:5mg/L CB处理4h能够显著提高猪孤雌囊胚的卵裂率和囊胚率(P0.05),减少囊胚细胞凋亡数量;DNA甲基转移酶(Dnmt1,Dnmt3a,Dnmt3b)、组蛋白乙酰转移酶(Hat1)、组蛋白去乙酰化酶(Hdac1)以及凋亡相关基因(Bax,Casp3)mRNA水平均明显下降;AcH3K9的表达无明显变化;而5hmc/5mc和H3K9me3的表达明显升高。该结果为进一步研究猪胚胎发育过程中的表观遗传变化提供了试验依据。     

Scriptaid联合5-Aza-CdR对水牛成纤维细胞DNA甲基化和组蛋白乙酰化修饰的影响

旨在探讨Scriptaid和5-Aza-CdR共同处理水牛耳部成纤维细胞(buffalo adult fibroblasts,AFs)对细胞生长特性、DNA甲基化和组蛋白乙酰化的影响,为提高水牛体细胞重编程效率提供一定的理论基础。首先使用不同浓度的Scriptaid(0,0.05,0.10μmol/L)和5-Aza-CdR(0,0.02,0.10μmol/L)分别处理AFs,筛选出最佳处理浓度后联合处理AFs 24h。随后采用免疫荧光染色技术分析联合处理后细胞的DNA甲基化和组蛋白H3K18乙酰化变化情况,并使用实时荧光定量PCR(QPCR)技术对其甲基化和乙酰化相关基因的表达进行检测。结果发现,适宜浓度的Scriptaid(0.05μmol/L)和5-Aza-CdR(0.02μmol/L)对细胞正常的形态、生长无显著影响。与对照组相比,联合处理后AFs中DNA甲基化水平显著降低,而组蛋白H3K18位点的乙酰化水平显著上升(P0.05)。QPCR分析结果显示,联合处理后AFs中的DNA甲基化相关基因Dnmt1、TET1和TET2的表达水平均出现下调,其中Dnmt1的表达水平显著低于对照组(P0.05);组蛋白去乙酰化酶HDAC1和HDAC2的表达水平也出现下调,其中HDAC1下调显著(P0.05);而组蛋白乙酰化转移酶CBP,P300和HAT1的表达水平出现上升,其中P300和HAT1的表达水平显著高于对照组(P0.05)。结果表明,适宜浓度的Scriptaid(0.05μmol/L)和5-Aza-CdR(0.02μmol/L)联合处理AFs,对细胞形态和生长无显著影响,但可以有效降低细胞甲基化水平并提高组蛋白H3K18位点的乙酰化水平。     

3种来源猪胚胎激活后6h内基因组DNA甲基化变化模式

主要探讨猪手工克隆(HMC)胚胎和体外受精(IVF)胚胎及孤雌激活(PA)早期胚胎发育过程中6 h内的单细胞核全基因组DNA甲基化模式.运用抗5-甲基胞嘧啶抗体免疫荧光和激光共聚焦显微镜检测相对荧先强度的方法,检测猪手工克隆胚在融合后6 h内全基因组DNA甲基化模式,并与同时期猪体外受精胚及孤雌激活胚胎进行比较,从而探讨这3种胚胎变化模式的异同.结果显示:3种不同来源的胚胎在培养后不同时间点比较时,2、4、6 h均呈强甲基化状态,且均呈下降趋势;在同时间点比较时,2 h和6 h这两个时间点检测到3种胚胎的相对荧光强度均差异不显著(P＞0.05),而在4 h时,HMC胚胎显著高于PA和IVF胚(P＜0.05).结论:猪手工克隆胚在融合后6h内虽然也发生类似IVF胚的全基因组DNA去甲基化,但其去甲基化不充分,甲基化程度偏高,这可能是导致核移植胚胎核重编程不充分、发育能力低的一个原因.     

Effect of DNA Methylation Inhibitor 5-Aza-CdR on Patterns of Pluripotent-related Genes and Methylation Genes in Handmade Cloning Embryo

The expression pattern of pluripotent gene Oct4 and Nanog,and methylation related genes Dnmt1 and Tets of handmade cloning (HMC) embryos were studied by Real-time quantitative PCR assay.The effect of genes expression pattern by 5-Aza-CdR on HMC reconstructed embryos was also explored. The results showed that the expression of Oct4,Nanog and Tet3 genes reached peak on 2-cell stage,the expression of Dnmt1 and Tet2 genes declined with the embryo development,while the expression of Tet1 gene increased.The use of 5-Aza-CdR didn't change the expression pattern of Oct4,Tet1 and Tet3 genes,but increased the expression of Nanog gene at the beginning of embryo development,while decreased the expression of Dnmt1 and Tet2 genes.The expression pattern of Oct4,Nanog,Dnmt1 and Tets genes in the development of HMC embryo was established,the use of 5-Aza-CdR could influence the methylation process of HMC embryo.     

Suberoylanilide hydroxamic acid,a novel histone deacetylase inhibitor,improves the development and acetylation level of miniature porcine handmade cloning embryos

Histone deacetylase inhibitors (HDACis) can change the histone acetylation and significantly enhance the developmental competence of the pre‐implantation SCNT embryo. To select a proper histone deacetylase inhibitor to improve the success rate and potentially developmental ability of handmade cloning (HMC) embryos of miniature porcine, we compared the effect of two histone deacetylase inhibitors (SAHA vs. VPA) on HMC embryo development, their histone acetylation level and the expression level of relevant genes. The blastocyst rate and number of blastocyst cells of HMC embryos treated with SAHA (SAHA‐HMC) or VPA (VPA‐HMC) were significantly higher than those of control (Control‐HMC), respectively, but there were no significant difference between SAHA‐HMC and VPA‐HMC groups. In addition, the acetylation level (AcH4K8) of Control‐HMC and VPA‐HMC embryos at the blastocyst stage, respectively, was significantly lower than that of in vitro fertilized (IVF) and SAHA‐HMC embryos. However, the acetylation H4K8 of the blastocysts had no significant difference between SAHA‐HMC and the IVF groups. The SAHA‐HMC blastocysts indicated comparative expression levels of Oct4 and HDAC1 (histone deacetyltransferase gene) with those of IVF blastocysts. In contrast, the expression levels of Oct4 were lower and those of HDAC1 were higher in the VPA‐HMC and Control‐HMC blastocysts, respectively, compared to those of the IVF blastocysts. Our results demonstrated that the HMC embryos treated by SAHA could promote the pre‐implantation development and increase the levels of histone H4K8 acetylation and the expression of the OCT4 gene, yet decrease the expression of the HDAC1 gene to the comparable level of the IVF embryos. Our results proved that SAHA may be a better histone deacetylase inhibitor for porcine HMC compared to VPA, and furthermore, it may indicate that SAHA can effectively correct the abnormal histone acetylation during the HMC embryo development and subsequently improve the full‐term developmental potential of the HMC embryos after embryo transplantation.     

牛印记基因的研究进展

基因组印记是一种表观调控机制,在哺乳动物的发育中具有重要作用。印记基因是仅一方亲本来源的同源基因表达,而来自另一亲本不表达的一种基因。近年来,印记基因被广泛研究。印记基因分为父系印记基因和母系印记基因,其表达具有组织特异性,而且在胚胎不同发育阶段的表达也具有一定差异,胚胎期的营养水平也影响印记基因的表达。DNA甲基化在调控印记基因的表达中起重要作用,影响细胞核移植过程细胞的表观重编程,并影响胎盘及内脏器官的正常发育;基因印记模式的改变也可以引起甲基化的改变进而导致基因印记的丢失等。本文综述了近年来关于牛的印记基因的研究进展情况,为印记基因的后续相关研究工作提供借鉴。     

The Dnmt3b splice variant is specifically expressed in in vitro-manipulated blastocysts and their derivative ES cells

Manipulation of preimplantation embryos in vitro, such as in vitro fertilization (IVF), in vitro culture (IVC), intracytoplasmic sperm injection (ICSI), somatic cell nuclear transfer (SCNT) and other assisted reproduction technologies (ART), has contributed to the development of infertility treatment and new animal reproduction methods. However, such embryos often exhibit abnormal DNA methylation patterns in imprinted genes and centromeric satellite repeats. These DNA methylation patterns are established and maintained by three DNA methyltransferases: Dnmt1, Dnmt3a and Dnmt3b. Dnmt3b is responsible for the creation of methylation patterns during the early stage of embryogenesis and consists of many alternative splice variants that affect methylation activity; nevertheless, the roles of these variants have not yet been identified. In this study, we found an alternatively spliced variant of Dnmt3b lacking exon 6 (Dnmt3bΔ6) that is specific to mouse IVC embryos. Dnmt3bΔ6 also showed prominent expression in embryonic stem (ES) cells derived from in vitro manipulated embryos. Interestingly, IVC blastocysts were hypomethylated in centromeric satellite repeat regions that could be susceptible to methylation by Dnmt3b. In vitro methylation activity assays showed that Dnmt3bΔ6 had lower activity than normal Dnmt3b. Our findings suggest that Dnmt3bΔ6 could induce a hypomethylation status especially in in vitro manipulated embryos.     

DNA甲基化对早期胚胎发育的影响

综合分析了DNA甲基化的表观遗传特征,结合DNA甲基化在不同物种、不同发育阶段胚胎中的调控模式,以期从早期胚胎死亡角度揭示DNA甲基化作用对胚胎早期发育基因的表达调控作用,进而阐明胚胎发育过程中的表观遗传调控机制。     

Effects of cumulus cells on in vitro maturation of oocytes and development of cloned embryos in the pig

The purpose of this study was to investigate the role of porcine cumulus cells (CC) in oocyte maturation and somatic cell nuclear transfer (SCNT) embryo development in vitro. Denuded pig oocytes were co-cultured with CC or routinely cultured in maturation medium without a feeder layer. Porcine CC inactivated with mitomycin C or non-inactivated were used for the feeder layer in co-culture with porcine SCNT embryos to investigate comparatively the developmental competence of cloned embryos. The DNA damage aspects of apoptosis and expression pattern of genes implicated in apoptosis (Fas/FasL) as well as the mRNA expression of DNA methyltransferase (Dnmt1, Dnmt3a) of porcine SCNT embryos were also evaluated by comet assay or real-time RT-PCR, respectively. The results showed that co-culture with CC improved the extrusion rate of pbI (49.3% vs 31.5%, p<0.05) and survival rate (75.7% vs 53.3%, p<0.05) of denuded oocytes, but had no effects on blastocyst developmental rate or 2-cell-stage survival rate of in vitro fertilization embryos. Co-culture with CC inactivated by mitomycin C improved the blastocyst developmental rate (26.6% vs 13.0%, p<0.05) and decreased the apoptotic incidence (27.6% vs 46.2%, p<0.05) of porcine cloned embryos. Co-culture with inactivated CC reduced Fas and FasL mRNA expression of cloned embryos at the blastocyst stage compared with NT controls (p<0.05), but there were no differences in Dnmt1 and Dnmt3a mRNA expression among groups. Co-culture with inactivated cumulus cell monolayer significantly increased blastocyst formation and decreased the apoptotic incidence in porcine cloned embryos during in vitro development.     

单胚胎RT-qPCR对猪体细胞核移植囊胚全能性的分析

为了检测猪体细胞核移植囊胚的质量和全能性基因表达量,采用体细胞核移植技术制备克隆胚胎并于体外培养5 d后获得囊胚,对照组为从人工授精5 d后的长白母猪体内获取的体内囊胚。在高倍镜下检测两组囊胚的形态,用Hoechest 33342染色细胞核DNA,记录囊胚细胞总数;建立单胚胎cDNA的制备方法,并用qPCR检测囊胚中全能性基因(Oct4、Nanog、Sox2)的表达量。结果表明,与体内囊胚相比,猪体细胞核移植囊胚质量较差,细胞数目显著降低(分别为110±10.3和54±12.6);并且全能性基因表达量显著下降(P<0.05)。由此可见,全能性基因表达量偏低是影响猪体细胞核移植囊胚发育能力的因素之一。     

羊水来源干细胞基因组特征的研究进展

人和动物存在羊水来源的多潜能干细胞(amniotic fluid-derived stem cells,AFSCs)。AFSCs被公认为是介于胚胎干细胞和成体干细胞之间的一种特殊的细胞类型,因此,AFSCs同时具有成体干细胞和胚胎干细胞的生物学特性。AFSCs干细胞特性的保持是以其标志基因以及基因组的整体特异表达为基础。目前,已经有相关研究对不同胎龄时期的AFSCs的标志基因Oct4、Sox2、Nanog、SSEA系列以及Tra-1-60和Tra-1-81等在基因组中的表达特征进行了分析。对AFSCs的相关研究进展进行综述,以期为羊水干细胞的进一步系统、深入研究提供理论依据。