小鼠单个植入前胚胎SSH方法的可行性

为建立一种能够一次性分离任意两个植入前胚胎之间全部的差别表达的基因的方法 ,在已有的单胚胎操作技术的基础之上 ,对单个植入前胚胎抑制性消减杂交 (singlepreimplantationembryosuppressionsubtractivehybridization ,SPE SSH)方法进行了初步的探索 ,分离到OM2和MⅡ d 2的基因片段 ,经GenBank和文献检索发现 ,这两个基因具有在MⅡ期和 2细胞期特异性表达的特点 .利用cDNA阵列所进行的鉴定也获得了同样的结果 ,而且所使用的材料极少 ,说明SPE SSH是一种强有力的分离和识别早期发育相关基因片段的实验技术 .若与单个卵裂球分离技术相结合 ,还可用于分离和识别人类早期分子诊断的标记性基因     

Effect of homologous peptides of differentiation factors HLDF and PEDF on preimplantation development of mice in vitro

We studied the effect of synthetic peptides PEDF-6 and HLDF-6 on preimplantation development of mouse embryos in vitro. PEDF-6 peptide corresponds to fragment 351-356 and of pigment epithelium-derived differentiation factor (PEDF), while HLDF-6 peptide corresponds to fragment 84-89 of differentiation factor HLDF isolated from HL-60 cell line. Despite high homology, these peptides had different effects on the early development. PEDF-6 had no effect on the cleavage of 2-4-cell embryos but decelerated blastocyst formation from such embryos and disturbed their structure. In the presence of HLDF-6 the blastomeres divided more actively as compared to the control and a higher number of embryos developed to the blastocyst stage. The effects of both peptides were stage-specific: the affect the embryos at early cleavage stages and, apparently, determine their further development at that moment although do not directly affect formation of the blastocysts.     

Effects of Growth Factors FGF2 and IGF2 on the Development of Parthenogenetic Mouse Embryos in utero and in vitro

We studied the effects of fibroblast growth factor 2 (FGF2) and insulin-like growth factor 2 (IGF2) on the development of parthenogenetic mouse embryos (CBA × C57BK/6)FF₁. The parthenogenetic embryos were treated in vitro during the preimplantation period and, at the blastocyst stage, transplanted into the uterus of pseudopregnant females. The addition of FGF2 at an optimal dose (2.5 ng/ml) to the culture medium increased twofold the number of embryos developed in utero to the somite stages as compared to the control: 18 and 43%, respectively. The parthenogenetic embryos (18–21 somites), treated and nontreated with FGF2 during the preimplantation period, were explanted for further development in vitro and treated with IGF2 at 2.5 g/ml. As a result, many more parthenogenetic embryos (> 87%) of both groups developed in vitro to the stage of 30 or more somites as compared to the control (59%). More than a half of FGF-2-treated parthenogenetic embryos developed to the stage of 40 and some of them, to the stage of 50 somites. The treatment of the parthenogenetic embryos with FGF2 alone at the preimplantation stages did not improve their development in vitro at the postimplantation stages. The results we obtained suggest that the treatment of parthenogenetic embryosin vitro with FGF2 during the preimplantation period increased twofold the number of somite embryos in utero, while their subsequent treatmentin vitro with IGF2 leads to a significant prolongation of their development, as compared to the control.     

Investigation of the Effect of Various Buffer Solutions Used for Microinjection of Gene-Engineering Constructs on Preimplantation Development of Mouse Embryos

We studied the effects of some buffer solutions used for microinjection in mammalian zygotes on preimplantation development of (CBA × C57BL) F₁ mouse embryos in vitro. The rate of embryo survival was estimated according to their capacity to develop to the stages of blastocyst and blastocyst hatched from zona pellucida. The results obtained suggested a reduced rate of survival of zygotes to the blastocyst stage after the injection into a pronucleus of the buffers Tris-HCl with EDTA, Tris-HCl with MgCl₂ and NaCl, and medium M2 (p < 0.05) and to the stage of blastocyst hatched from zone pellucida after injection of a Dulbecco solution, as compared to the control. No differences were found in the survival rate of zygotes injected with different buffer solutions.     

Leukemia Inhibitory Factor (LIF) Improves and Prolongs the Development of Parthenogenetic Mouse Embryos

We studied the effect of the growth factor LIF on the development of parthenogenetic mouse embryos (CBA × C57BL/6)F1. LIF was added to the culture medium at 10, 50, 100, and 250 ng/ml at the morula stage and parthenogenetic embryos were cultured in vitro until the late blastocyst stage and then transplanted in the uterus of pseudopregnant females, which were then sacrificed on day 12 of pregnancy. All the LIF doses used improved the development of parthenogenetic mouse embryos at the preimplantation stages and increased the amount of blastocysts by 15%, on average, as compared to the control. LIF at 50 and 100 ng/ml increased approximately twice the number of embryos that reached the somite stages. Some of them reached the stage of 32–45 somites and had fore and hind limb buds. No such embryos were found in the control. Well formed placenta was observed in 6% of the embryos treated with LIF and the most pronounced effect was recorded at 100 ng/ml. The data we obtained suggest that exogenous LIF can improve pre- and postimplantation development of parthenogenetic mouse embryos due, possibly, to increased survival rate of embryonic stem cells derived from the inner cell mass of blastocysts. LIF improves not only the development of the parthenogenetic embryoper se, but also the formation of its extraembryonic envelopes, which leads to the development of a larger placenta in LIF-treated parthenogenetic embryos, as compared to the control.     

小鼠母源因子对早期胚胎发育的影响

在脊椎动物中发育过程中,卵母细胞要经历MII期停滞、受精、早期胚胎发育的启动、胚胎基因组的转录激活、并指导完成个体的发育过程。同时,核移植过程中,分化的细胞核在去核的卵母细胞中能够重编程到胚胎早期的状态并能完成个体的发育过程。在这些发育过程中母源因子都发挥了极其的重要作用。在小鼠胚胎发育研究中发现,小鼠的基因组激活发生在2细胞期,这一时期标志着合子的发育由卵母细胞控制向胚胎控制的过渡,期间发生一系列复杂的生化过程。体外培养的小鼠的胚胎的发育阻断也易发生的2细胞时期。因此对卵母细胞及早期胚胎母源因子的研究,将有利于了解早期体外培养胚胎和克隆胚胎发育失败的原因,为提高体外培养和克隆胚胎发育的成功率提供理论的基础。     

小鼠体内外受精植入前胚胎全基因组甲基化模式比较

为考察体外受精、操作及培养环境对体外受精的小鼠植入前胚胎全基因组DNA甲基化模式的影响,本研究以体内受精的植入前胚胎作为对照,采用间接免疫荧光法检测小鼠体内外受精植入前胚胎基因组DNA甲基化模式.实验结果表明,体外受精各期植入前胚胎呈现出与之相应时期的体内受精植入前胚胎不同的DNA甲基化模式和水平,原核期甲基化水平较高,2-4-、8-细胞期明显降低,而桑葚胚和囊胚期又略有升高.各期体外受精植入前胚胎的基因组DNA甲基化水平都比同时期体内受精胚胎的甲基化水平低.本实验结果部分显示了体外受精、操作及培养环境可能对正常的DNA甲基化模式产生影响,造成体外受精植入前胚胎甲基化模式异常.     

乙醇和液体石蜡对早期小鼠胚胎体外发育的影响

哺乳动物早期胚胎体外培养技术是研究早期胚胎发育和胚胎工程的基本手段。目前最常用的方法是采用液体石蜡覆盖的液滴培养法。该方法中所用液体石蜡和ＣＯ２质量的好坏对体外培养胚胎的发育有严重影响。本文就此对几种中国产液体石蜡和含气体乙醇的ＣＯ２作为胚胎体外培养条件对胚胎发育的影响进行了研究。取成熟小鼠受精后的２细胞和８细胞胚胎分别用于两组实验。一、不同品牌液体石蜡对胚胎发育的影响。体外培养采用液体石蜡覆盖液滴培养法。所用液体石蜡均经过水洗。ＣＯ２为经过一次水滤的工厂粗制品,其乙醇含量经气相色谱仪测定约为０．１８％。二、不同浓度乙醇对胚胎发育的影响。采用试管培养法,采用乙醇浓度不同的培养液。表１为五个不同厂家的液体石蜡对早期胚胎体外培养的影响。其中,Ⅰ、Ⅱ（上海、北京）号两种产品符合胚胎体外培养的要求,可使２细胞后期胚胎发育到囊胚的比率达到９２％以上,对细胞没有毒害作用（图１Ａ和图２Ａ、Ｂ）。其余,则不符合要求,对细胞有毒害作用（图１Ｂ）,甚至用无水乙醇和水先后各洗涤３至４遍,亦无改善。由于影响液体石蜡质量的硝基萘可溶于乙醇而被清除,说明这些不合格的液体石蜡中可能还含有其它对胚胎发育有毒害作用的未知因素,有待进一步查明     

Genomic Imprinting and Problem of Parthenogenesis in Mammals

Genomic imprinting belongs by its nature to problems of epigenetics, which studies hereditary changes in gene expression not related to defective sequences of DNA nucleotides. Epigenetic mechanisms of control, including genomic imprinting, are involved in many processes of normal and pathological development of humans and animals. Disturbances of genomic imprinting may lead to various consequences, such as formation of developmental anomalies and syndromes in humans, appearance of the large offspring syndrome and increased mortality upon cloning of mammals, and death of parthenogenetic embryos soon after implantation and beginning of organogenesis. The death of diploid parthenogenetic or androgenetic mammalian embryos is determined by the absence of expression of the genes of imprinted loci of the maternal or paternal genome, which leads to significant defects in development of tissues and organs. A review is provided of the studies aimed at search of possible normalization of misbalanced gene activity and modulation of genomic imprinting effects during parthenogenetic development in mammals.__________Translated from Ontogenez, Vol. 36, No. 4, 2005, pp. 300–309.Original Russian Text Copyright © 2005 by Platonov.     

猪早期孤雌激活胚不同发育阶段差异基因表达的研究

收集2细胞、4细胞、8-16细胞时期的猪孤雌激活胚,采用SPEDDRT-PCR方法挑选不同时期的差异表达产物,通过反向northern杂交去除假阳性的条带。将阳性条带克隆入T载体中,经过PCR鉴定后挑选其中的阳性克隆进行测序,筛选了8个代表不同时期表达差异的cDNA片段,编号为DD1-DD8。经过与GenBank中的数据进行同源性分析,发现其中DD1和DD2没有相似的数据, 提交数据库获得GenBank登录号(EU545158, EU545159);其余的DD3-DD8发现了相似性较高的数据,但除DD3外均无基因功能说明需要进行进一步的研究。     

小鼠心脏发育的形态学研究

目的建立小鼠心脏正常发育的时间表以及对应的形态学特征模式.方法小鼠胚胎ED8.5、ED9.5、ED10.5、ED11.5、ED12.5、ED14.5、ED16.5、ED18.5和P1(postnatal day)(出生后1 d的仔鼠)标本,进行整体或心脏部位不同轴向切片,HE染色,采用PCTV图像分析系统,对各时相小鼠心脏形态发育特征进行研究.结果 ⑴细胞结构发育的时空模式:① ED8.5时,生心板形成;ED9.5时,心肌细胞呈不规则的纺锤形,细胞的大小多样化,细胞核小;ED10.5时,小血管和着色较浅的肌原纤维出现,细胞之间连接较松;ED11.5时,心肌纤维排列较紧,纵断面上呈细长形,横断面上呈不规则多角形;ED12.5时,细胞核着色更清晰,心肌细胞形状逐渐规则,细胞之间紧密连接,同时闰盘结构出现在心室心肌细胞.②ED12.5时心肌小梁结构第一次在心室出现,ED14.5时增厚,而在心房少见心肌小梁.⑵心室结构的形成和心脏发育的成熟:①心肌间充质网络结构在ED10.5的心室中明显呈现,随着它的发育,心室的心内膜在ED11.5出现,心室心外膜可以辨认.②房室隔在ED12.5完全形成,心内膜垫在ED12.5开始发生并快速发育,促进室间隔在ED14.5完全形成.③心包膜在ED16.5可明显辨认,心包膜腔形成,此时近段流出道心内膜垫完全心肌细胞替换.结论肌原纤维细胞和心肌间充质细胞同时在ED10.5出现,提示肌原纤维对心肌细胞的成型和心肌化起作用.细胞的结构变化和心肌层的成熟过程,显示小鼠心脏部位成熟时间的不同,心室成熟相对较晚.     

Preimplantation embryonic development of spontaneous mouse parthenotes after oocyte meiotic maturation in vitro

Of eggs ovulated in LT/Sv mice, 10–20% undergo spontaneous parthenogenetic activation, and 40–50% of the parthenotes develop to blastocysts when cultured in simple defined medium from the one-cell stage. Similar percentages of oocytes isolated from Graafian follicles undergo parthenogenetic activation after spontaneous maturation in simple defined medium, but embryonic development proceeds no further than the two-cell stage. The simple defined medium that supported preimplantation development of ovulated eggs and spontaneous maturation of extrafollicular oocytes contained no serum, free amino acids, or vitamins. The present experiments were conducted to determine what conditions during spontaneous maturation of extrafollicular oocytes could promote the ability of oocytes to develop to blastocysts after parthenogenetic activation and mimic the environment of preovulatory follicles. Cumulus-enclosed oocytes that were matured in simple medium supplemented with fetal bovine serum (FBS) developed to blastocysts after spontaneous parthenogenetic activation. Furthermore, minimum essential medium (MEM), a complex medium containing free amino acids and vitamins, could substitute completely for FBS for maturing oocytes from (C57BL/6J × LT/Sv)F₁ mice, and to a lesser extent for maturing LT/Sv oocytes. Therefore, even though germinal vesicle breakdown in mouse oocytes and preimplantation development of mouse eggs can occur in the absence of an exogenous supply of free amino acids and vitamins, a complete, or normal, mouse oocyte maturation cannot. These results also demonstrated that gonadotropins are not necessary during oocyte meiotic maturation for parthenogenetically activated eggs to develop through the preimplantation stages. Luteinizing hormone or 17β-estradiol in MEM during oocyte maturation had no effect on the subsequent development of parthenotes. In contrast, follicle stimulating hormone (FSH) and progesterone in the maturation medium decreased the number of ova that subsequently cleaved, and FSH decreased the number of cleaved eggs that developed to blastocysts.     

异源Oct-4基因启动子在猪、兔和小鼠早期胚胎中的时空表达活性

Oct-4是一种哺乳动物早期胚胎中特异表达的转录因子,它与细胞多能性的维持有关．异源Oct-4基因在早期胚胎中的表达模式尚不明确．构建了一个以完整的牛Oct-4调控区指导GFP表达的转基因结构pOct-4(p)-GFP,通过单精子注射的方法将其导入猪、兔和小鼠的受精卵中,分析其在胚胎发育过程中的表达情况．结果显示：牛Oct-4启动子驱动的GFP基因在3个物种的2-细胞胚胎就已经开始表达,在囊胚期表达加强且只特异表达于内细胞团中,而不表达于滋养层．研究表明：牛的Oct-4启动子在其他物种中也具有表达活性,异源性Oct-4启动子在不同物种的早期胚胎中具有相似的表达模式．     

The effects of bovine serum albumin, bovine uterine fluid, and heat-treated bovine serum on in vitro mouse embryo development

Two-cell mouse embryos were cultured in Whitten's medium with one of three supplements: bovine serum albumin (WM + BSA), heat-treated bovine serum (WM + HTBS) or bovine uterine fluid (WM + BUF). Protein concentrations for cultures of WM + BSA were 50.2, 100.5, 251.2, 502.5, and 1005.0 mug/ml and for WM + HTBS were 70.4, 105.1, 269.0, 524.5 and 1193.9 mug/ml. Protein concentrations ranged from 56.9 to 739.1 mug/ml for 22 WM + BUF samples. Embryo development in all media was significantly correlated with the log total protein concentration. When compared to WM + BSA, development was not significantly inhibited or stimulated in any WM + BUF cultures or in WM with 70.4, 524.5 and 1193.9 mug/ml HTBS. Development was enhanced in WM with 105.1 and 269.0 mug/ml HTBS (P<0.05). The results suggest that at the protein concentrations used, culture media supplemented with BUF and BSA support similar mouse embryo development. Culture medium supplemented with HTBS supported embryo development more than medium with BSA. Uterine factors in the bovine capable of enhancing or inhibiting early embryo development were not detected.     

LH与hCG对昆明小鼠卵母细胞体外成熟的影响

目的研究促黄体素（LH）、人绒毛膜促性腺激素（hCG）对昆明小鼠卵母细胞体外成熟的影响。方法小鼠经注射孕马血清促性腺激素（PMSG）48h后,摘取卵巢获得未成熟卵母细胞,分别在含不同浓度的LH和hCG的成熟液中,或将LH和hCG以不同的浓度组合加入到成熟液,进行体外成熟。结果经15.16h的成熟培养,5个浓度LH组中的极体率均高于对照组,其中200IU/mL组显著高于50IU/mL、400IU/mL、300IU/mL组和对照组（P〈0.05）;5个浓度hCG组的极体率与对照组极体率无显著差异（P〉0.05）;协同组中15IU/mL hCG＋200IU/mL LH组的极体率显著高于对照组和其它各处理组。结论LH对小鼠卵母细胞的体外成熟有一定的促进作用。     

雷帕霉素靶在小鼠受精卵早期发育中的作用研究

哺乳动物雷帕霉素靶(mTOR)是细胞生长的中心调控因子,应用RT-PCR、免疫印迹、放射性同位素体外测定酶活性等方法,研究mTOR在小鼠受精卵第一次有丝分裂过程中在卵中的表达、活性变化以及对卵裂的影响.研究发现mTOR在小鼠卵母细胞和受精卵中都有表达,在mRNA水平,mTOR从G2期开始降解,在蛋白水平,则各期没有明显变化;mTOR的激酶活性在受精后明显升高,并且在整个1-细胞期保持较高活性;mTOR的特异性抑制剂雷帕霉素能抑制卵裂,并且能抑制成熟促进因子MPF的调节亚基cyclin B的表达,从而抑制了MPF的活性.结果表明mTOR可能通过促进MPF的激活而促进小鼠受精卵的分裂.     

早产儿语言发展的影响因素及其干预对策

早产儿的语言发展受到多种因素的影响,可能导致他们在词汇、语法、语音等方面出现发展滞后或障碍。本文首先简述了影响早产儿语言发展的生物学和环境因素的研究进展,其中生物学因素包括早产程度、体重和性别、新生儿发病率和疾病严重程度等,环境因素则包括新生儿重症监护室的环境、家庭中的语言环境和社会因素等。在明确这些影响因素的基础上,本文强调了早期评估和早期干预是优化早产儿语言发育效果的关键步骤,并分析了具体的干预对策,例如生理和神经干预、优化新生儿重症监护室（NICU）环境、增强家庭语言互动、多学科合作和社会支持等。此综述旨在探讨影响早产儿语言发展的各种因素,并总结出有效的早期干预措施,为其提供更为全面的语言发展支持。     

Effect of co-culturing of half-destroyed and intact 4-cell mouse embryos in varying ratios on subsequent in vitro development

We studied the co-culturing effect of intact and half-destroyed 4-cell mouse embryos on blastocyst formation rate and cell counts. A laser beam was used to produce a hole and destroy an adjacent blastomere in two opposite areas of the zona in the experimental group (n = 342), and to open two opposite zonal holes in the controls (n = 318). Control and half-destroyed embryos were cultured together in varying ratios of 10:0, 7:3, 5:5, 3:7, and 0:10 (group 1-5, respectively) for 48 h in 10 μl drops of cleavage medium. They were then separated and cultured in blastocyst medium for 24 h. The results showed that half-destroyed embryos had no effect on the blastulation rates of controls (97-100%, P = 0.28). Neither was there a difference in the number of ICM (27.3 ± 6.7, 29.4 ± 9.9, 27.7 ± 9.3, 26.5 ± 6.4, in group 1-4, respectively; P = 0.491), TE (47.7 ± 18.6, 52.3 ± 13.9, 48.4 ± 19.2, 57.3 ± 12.9, in group 1-4, respectively; P = 0.101), nor total cells (75.0 ± 19.5, 81.3 ± 17.1, 76.1 ± 19.6, 83.7 ± 16.2, in group 1-4, respectively; P = 0.188) in the resulting blastocysts. However, among half-destroyed embryos, cleavage arrest decreased (58.3%, 39.6%, 17.9%, and 8.3%, in group 5 to 2, respectively; P < 0.001) and blastocyst development increased (38.3%, 58.2%, 72.6%, and 88.9%, in group 5 to 2, respectively; P < 0.001) following co-culturing with intact controls. These embryos had a higher number of ICM cells (P = 0.035), but no significant changes in TE (P = 0.262) and total cell counts (P = 0.065). The findings indicate that the co-culturing of half-destroyed with intact embryos increased the blastulation rate of the first but had no effect on the latter.     

不同来源乳铁蛋白对幼鼠肠道发育的影响

目的 探究哺乳期乳铁蛋白（lactoferrin，LF）的缺失及不同来源LF补充后对幼鼠肠道发育的影响。方法 以LF基因敲除型雌鼠作为哺乳母鼠造成幼鼠哺乳期无LF的摄入，且从幼鼠出生第3~21天每日人工饲喂100 mg/kg 牛血清白蛋白（BSA）、牛源乳铁蛋白（bovine Lactoferrin，bLF）及重组人源乳铁蛋白（recombinant human Lactoferrin，rhLF），于幼鼠21日龄取样，测定各组小鼠小肠发育指标。结果 在本实验周期下，哺乳期rhLF的补充显著性增加小鼠回肠绒毛长度/隐窝深度值（P<0.05），且上调回肠Occludin和ZO-1基因的表达（P<0.05），增加小鼠十二指肠、空肠和回肠麦芽糖酶酶活/乳糖酶酶活比值（P<0.05），表明哺乳期rhLF的补充能够增强小鼠肠道消化吸收能力和肠屏障功能；哺乳期bLF的补充显著增加小鼠十二指肠及回肠麦芽糖酶活性/乳糖酶活性比值（P<0.05）。结论 对于哺乳期无LF摄入的乳鼠来说，哺乳期间LF的补充能够增强乳鼠肠道对营养物质的消化吸收能力、促进肠道的发育成熟、增强肠道屏障功能，并且，本实验中rhLF表现出比bLF更加有效的作用。     

Effect of oocyte vitrification on DNA damage in metaphase II oocytes and the resulting preimplantation embryos

As an assisted reproduction technology, vitrification has been widely used for oocyte and embryo cryopreservation. Many studies have indicated that vitrification affects ultrastructure, gene expression, and epigenetic status. However, it is still controversial whether oocyte vitrification could induce DNA damage in metaphase II (MII) oocytes and the resulting early embryos. This study determined whether mouse oocytes vitrification induce DNA damage in MII oocytes and the resulting preimplantation embryos, and causes for vitrification‐induced DNA damage. The effects of oocyte vitrification on reactive oxygen species (ROS) levels, γ‐H2AX accumulation, apoptosis, early embryonic development, and the expression of DNA damage‐related genes in early embryos derived by in vitro fertilization were examined. The results indicated that vitrification significantly increased the number of γ‐H2AX foci in zygotes and two‐cell embryos. Trp53bp1 was upregulated in zygotes, two‐cell embryos and four‐cell embryos in the vitrified group, and Brca1 was increased in two‐cell embryos after vitrification. Vitrification also increased the ROS levels in MII oocytes, zygotes, and two‐cell embryos and the apoptotic rate in blastocysts. Resveratrol (3,5,4′‐trihydroxystilbene) treatment decreased the ROS levels and the accumulation of γ‐H2AX foci in zygotes and two‐cell embryos and the apoptotic rate in blastocysts after vitrification. Overall, vitrification‐induced abnormal ROS generation, γ‐H2AX accumulation, an increase in the apoptotic rate and the disruption of early embryonic development. Resveratrol treatment could decrease ROS levels, γ‐H2AX accumulation, and the apoptotic rate and improve early embryonic development. Vitrification‐associated γ‐H2AX accumulation is at least partially due to abnormal ROS generation.