哺乳动物子宫自然杀伤(uNK)细胞对妊娠的调控作用

子宫自然杀伤(uterine natural killer, uNK)细胞是哺乳动物妊娠早期胎盘蜕膜中数量最多的免疫细胞。妊娠期间,uNK细胞对子宫免疫耐受环境建立,胎盘以及胎儿的发育等均具有重要调控作用。目前,有关妊娠中uNK细胞独特生物活性调控机制研究非常缺乏。本文综合相关研究,介绍了妊娠过程中uNK细胞在母-胎免疫耐受建立、胎盘和胎儿发育过程中重要调控作用,并总结激素、糖代谢以及DNA甲基化等对其他组织NK细胞调控机制研究,为探索uNK细胞调控分子机制提供新的研究方向和思路。     

Elevated Histone H1 (MPF) and Mitogen-activated Protein Kinase Activities in Pig Oocytes Following In Vitro Maturation do not Indicate Cytoplasmic Maturation

Effects of different media (TCM 199 + BSA, TCM 199 + FCS, TCM 199 + NBCS, Whitten's medium + BSA) supplemented with estradiol-17β and two isolated and everted follicle shells on MPF and MAP kinase activities and the sensitivity to parthenogenetic activation of pig oocytes were examined at the end of culture (48 h). Elevated ( P  <   0.05) activities of MAP kinase were recorded in metaphase II oocytes following culture in Whitten's medium, whereas MPF levels were lowest ( P  <   0.05) in MII oocytes matured in TCM 199 supplemented with BSA. Oocytes matured in TCM 199 based media showed higher ( P  <   0.05) activation rates when compared to oocytes incubated in Whitten's medium. Whitten's medium supplemented with different protein sources (amino acids, FCS, BSA) was used to study the effects of different exposure periods to eCG/hCG stimulation on MPF and MAP kinase activities and in vivo fertilisability following culture for 48 h. MPF and MAP kinase activities were significantly increased by eCG/hCG stimulation of COCs during maturation. Further, the continuous presence of eCG/hCG during culture (48 h) significantly increased the levels of both kinases in comparison to stimulation by gonadotrophins alone during the first 24 h of incubation. In vivo fertilisation of oocytes matured in Whitten's medium supplemented with eCG/hCG for 24 or 48 h led to a significant retardation of early embryonic development compared to ovulated oocytes. In conclusion, media composition and gonadotrophin stimulation affect MPF/MAP kinase activities and the susceptibility to parthenogenetic activation of IVM oocytes. However, elevated kinase levels in pig oocytes following culture do not indicate complete cytoplasmic maturation.     

哺乳动物细胞核移植研究进展

哺乳动物细胞核移植(Nuclear transfer,NT)是目前生物技术领域研究的热点之一,其科学意义和应用价值重大。本文根据近年来国内外的研究进展,对核移植技术研究概况、目前存在的问题和应用前景作一综述,希望能够对从事核移植研究的同行有所帮助。     

干细胞因子及其受体系统在哺乳动物生殖细胞发育过程中的作用

干细胞因子(Stem cell factor,SCF)及其受体(c—kit)自1990年被发现以来,已经逐渐受到科学工作者的重视。现在,对干细胞因子及其受体的研究,特别是该系统在发育调控中的作用,已成为研究的热点。笔者就SCF、c—kit系统在哺乳动物生殖细胞发育中的作用及研究现状作以简要的综述。     

表皮生长因子对水牛卵母细胞体外培养核质成熟的影响

为了探讨表皮生长因子（EGF）对水牛卵泡卵母细胞体外培养核质成熟的影响，在以TCM199为基础的成熟液中加入不同浓度的EGF（0、10、25、50、100 ng/ml），体外成熟培养24~26 h，观察第一极体（PB1）的排放；随后进行孤雌激活检测其分裂率、囊胚发育率、囊胚孵化率，并用Hoechst33342染色后计算囊胚的细胞数。结果发现添加EGF各组的卵母细胞第一极体排放率显著提高（P<0.05）；成熟液中添加25 ng/ml EGF时，卵裂率及囊胚发育率（分别为80.0%、44.8%）明显高于对照组（分别为69.3%、31.9%，P<0.05），但对囊胚的细胞数影响不大。EGF不仅促进水牛卵母细胞体外培养的核成熟，而且有利于卵母细胞的胞质成熟，其中EGF的最佳浓度为25 ng/ml。     

卵丘细胞在卵母细胞体外成熟培养过程中的作用

介绍了卵丘细胞在卵母细胞体外成熟培养过程中的作用,主要从卵丘细胞与卵母细胞的结构与功能联系,卵丘扩展对卵母细胞体外成熟的影响及卵丘细胞层数对卵母细胞体外成熟的影响三个方面加以阐述。     

草鱼→鲤的细胞核移植

用草鱼的头肾组织细胞核、草鱼的头肾细胞核与鲤鱼成熟去核卵之杂种胚胎细胞核、草鱼的囊胚细胞核,分别与鲤鱼的去核卵细胞质配合进行核移植操作.结果,体细胞核移植,在亚科间的组合——草鱼核 鲤质中获得原肠期胚胎,得率为1%;体细胞继代核移植,在草鱼核 鲤质中获得胚孔封闭期胚胎,得率为5%;胚胎细胞核移植,在草鱼核 鲤质中获得眼球色素出现期胚胎,得率为2%.草鱼→鲤核-质杂种胚胎的胎盘形状、大小与鲤鱼的相似,而大于草鱼;胚盘细胞数量也多于草鱼胚胎同期胚盘,而类似于鲤鱼同期胚盘.     

snRNA在哺乳动物中基因表达研究进展

小核RNA(snRNA)是真核生物转录后加工过程中RNA剪接体的主要成分,参与mRNA前体的加工过程。通过调控哺乳动物基因表达的能力为基因治疗癌症和神经退行性等疾病提供了新的理论依据。本文就snRNAs的分类、功能、基因表达染色质结构以及转录后修饰进行简要概述,为哺乳动物后续的相关研究提供参考。     

哺乳动物核移植研究进展

哺乳动物核移植(Nuclear Transplantation,NT)是目前生物技术领域研究的热点之一,其科学意义和应用价值重大。本文根据近年来国内外的研究进展,对核移植技术步骤、核移植研究概况及目前核移植研究中存在的问题作一综述,希望能够对从事核移植研究的同行有所帮助。     

In Vitro Maturation of Dromedary (Camelus dromedarius) Oocytes: Effect of Different Protein Supplementations and Epidermal Growth Factor*

The present experiment was aimed to compare the effect of different protein supplementation sources, foetal calf serum (FCS), oestrous dromedary serum (EDS) and BSA, in experiment 1, and the effect of different concentrations of epidermal growth factor (EGF), in experiment 2, on in vitro nuclear maturation of the dromedary oocytes. Cumulus oocyte complexes (COCs) were harvested from the ovaries collected from a local slaughterhouse by aspirating the visible follicles in PBS supplemented with 5% FCS. Pooled COCs were randomly distributed to 4‐well culture plates containing 500 μl of the maturation medium and cultured at 38.5°C in an atmosphere of 5% CO₂ in air for 32–36 h. The basic maturation medium consisted of TCM‐199 supplemented with 0.1 mg/ml L‐glutamine, 0.8 mg/ml sodium bicarbonate, 0.25 mg/ml pyruvate, 50 μg/ml gentamicin, 10 μg/ml bFSH, 10 μg/ml bLH and 1 μg/ml estradiol. In experiment 1, this medium was supplemented with 10% FCS, 10% EDS or 0.4% BSA, whereas in experiment 2, it was supplemented with 0.4% BSA and 0, 10, 20 or 50 ng/ml of EGF. The oocytes were fixed, stained with 1% aceto‐orcein stain and their nuclear status was evaluated. Oocytes were classified as germinal vesicle, diakinesis, metaphase‐I, anaphase‐I (A‐I), metaphase‐II (M‐II) and those with degenerated, fragmented, scattered, activated or without visible chromatin as others. There was no difference (p > 0.05) observed in the proportion of oocytes reaching M‐II stage between the media supplemented with FCS (71.5 ± 4.8), EDS (72.8 ± 2.9) and BSA (72.7 ± 6.2). In experiment 2, a higher proportion (p < 0.05) of oocytes reached M‐II stage when the medium was supplemented with 20 ng/ml of EGF (81.4 ± 3.2) when compared with the media supplemented with 10 ng/ml (66.9 ± 4.1) and control (67.2 ± 7.1) groups. It may be concluded that the maturation media for dromedary camel oocytes can be supplemented with any of the three protein sources, i.e. FCS, EDS and BSA without any significant differences on the maturation rates. Also, a supplementation of 20 ng/ml of EGF in the maturation medium seems to be optimal and improves the nuclear maturation of dromedary camel oocytes.     

Intermediate Filament Keratin Dynamics During Oocyte Maturation Requires Maturation/M‐Phase Promoting Factor and Mitogen‐Activated Protein Kinase Kinase Activities in the Hamster

Research related to intermediate filaments in mammalian oocytes remains poorly advanced. We investigated keratin reorganization in oocytes during meiotic maturation using immunofluorescence, and examined effects of inhibitors for cdc2 and mitogen‐activated protein kinase kinase (MAPKK) on keratin assembly. In germinal vesicle (GV) oocytes (n = 26), large and oval‐shaped aggregates of non‐fibrillar keratin were found in the cortical ooplasm (designated as a ‘cortical’ pattern). The delicate network of keratin filaments was concentrated in the GV periphery. The large keratin aggregates began to divide into small fragments at the pro‐MI/MI stage (n = 22, designated as a ‘fragmented’ pattern). Some keratin fragments were occasionally broken down into several granules at the peripheral region. In the MII oocytes (n = 24), the filament network was extended over the ooplasm and numerous keratin granules were scattered across the oocyte (designated as a ‘granular’ pattern). After 12 h of incubation with roscovitine, 66.7% of the oocytes (20/30) were at the GV stage and showed a cortical pattern of keratin. After incubation with U0126, most oocytes (83.9%, 26/31) were at the MII stage; most of them (76.9%, 20/26) showed a fragmented pattern of keratin. The increasing complexity of keratin filament network from the GV to MII stages suggests a possible role in maintaining cell integrity under physical stress after ovulation. In fact, maturation/M‐phase promoting factor is necessary for such keratin reorganization, as is meiotic nuclear progression. In addition, MAPKK is involved in keratin reorganization from a fragmented pattern to a granular pattern.     

Wound Healing: The Role of Platelet-Derived Growth Factor and Transforming Growth Factor Beta

Recent investigation into the mechanisms of wound healing has indicated the interaction of many substances, including several growth factors. The activity of platelet-derived growth factor (PDGF) and transforming growth factor beta (TGF-β), are best defined. Both factors are secreted primarily from the alpha granules of platelets, but also from activated macro-phages and fibroblasts. Investigation implicates the platelet as the initiator of wound healing, secreting PDGF, TGF-β, and other factors that are chemotactic for monocytes, macrophages, and fibroblasts. Although their mode of action and degree of effect are different, both PDGF and TGF-β increase the collagen content and early rate of gain of strength in wounds in normal and compromised tissue. In normal tissue, however, there is no long-term effect on wound outcome. The use of exogenous growth factors offers potential for chemical manipulation of the healing wound, particularly in tissues that are compromised, or where healing is abnormal.     

Effects of Low Oxygen Tension for in vitro Oocyte Maturation and Early Embryo Development through Hypoxia-inducible Factor (HIF)

Oocytes and early embryo in vitro culture (IVC) was a key technology of mammalian embryo engineering and was the basis of biotechnology such as genetically modified (GM) and cloning research.The factors,which affect the efficiency of in vitro culture for oocytes and early embryo,include the composition,ion concentration and osmotic pressure of the culture medium,and the gas composition and temperature of the culture environment. Recent studies have shown that the oxygen tension in the culture systems can significantly affect the development of embryos,and this effect is mainly regulated by the hypoxia-inducible factor (HIF). This paper reviewed the effects of the oxygen tension on oocyte maturation and early embryo development,and the structure and regulation mechanism of HIF were discussed,in order to provide a theoretical basis for further explore the application of hypoxia method in the in vitro culture of oocytes and early embryo.     

低氧分压通过低氧诱导因子(HIF)影响卵母细胞的体外成熟和早期胚胎的发育

卵母细胞和早期胚胎的体外培养(IVC)是哺乳动物胚胎工程的一项关键技术,是生殖生物学和转基因与克隆技术等生物技术研究的基础.影响卵母细胞和早期胚胎体外培养的因素包括培养液组成成分、离子浓度和渗透压,培养环境气相组成和温度等.最近研究表明,培养系统中的氧分压可以显著影响胚胎的发育,而这种影响主要由低氧诱导因子(hypoxia-inducible factor,HIF)进行调控.作者综述了氧分压及HIF对卵母细胞和早期胚胎体外发育的影响,并对HIF的结构及调控机制进行讨论,为研究低氧培养技术在卵母细胞和早期胚胎体外培养中的应用提供依据.     

A Potential Role for Platelet-activating Factor (PAF) During Bovine Placentation

Introduction:  Leg weakness causes high economic losses in commercial poultry. The unspecific term includes, amongst others, diseases of the skeletal system caused by genetic, nutritional or microbiological factors. The analysis of computed tomography images may be a new tool to discriminate and evaluate skeletal abnormalities in turkeys and can therefore provide valuable hints in the treatment of turkey stocks suffering from leg weakness.
Material and Methods:  Legs of healthy turkeys and those showing clinical signs of leg weakness were examined using a Tomoscan M/EG/Compact (Philips Medical Systems) CT system. The slice thickness and distance were set to 2 mm. In addition to the evaluation of the obtained images, the software system 3D-Doctor was used to create three-dimensional objects of the investigated samples.
Results:  Differences between physiological and abnormal parts of the leg bones are distinguishable in the CT-images and three-dimensional reconstructed objects. Bone density can be calculated and thus provides a base to estimate possible nutritional demands or deficiencies. The three-dimensional reconstruction compensates for the difficulties in understanding the detailed anatomical CT cross-sections by which the examiner is confronted while studying the CT images.
Conclusion:  Although computed tomography of poultry is too expensive for routine herd diagnosis, and far less for single-animal diagnosis, it is a valuable tool for investigating skeletal abnormalities and is very useful in examining samples related to leg weakness.     

Ghrelin在哺乳动物免疫系统中的作用研究进展

Ghrelin是一种具有多种生物学功能的生长激素释放肽,近年来其在哺乳动物免疫系统中的作用研究日益丰富,而在禽类免疫系统中的研究相对较少。论文就Ghrelin在调节哺乳动物淋巴细胞增殖、细胞因子分泌、炎症及组织损伤等方面的研究进展进行了综述,同时将禽类Ghrelin在免疫系统中的研究与之进行比较,为深入探索禽类Ghrelin在免疫系统中的功能及机制提供参考。     

FSH对绵羊卵母细胞体外核成熟的影响

为了探讨FSH对绵羊卵母细胞核成熟的影响,本试验将绵羊卵母细胞体外成熟24 h,并在成熟过程中的4个不同时间段内添加FSH,统计各个时间段卵母细胞的生发泡破裂(germinal vesicle break down, GVBD)及第一极体排出情况。结果显示：①在体外成熟培养的前4 h或前8 h添加FSH,经体外成熟的卵母细胞其生发泡破裂率与不添加FSH组无显著差异;②在体外成熟的4~24 h或8~24 h添加FSH,其第一极体排出率与不添加FSH组有极显著差异。说明,在本试验条件下,FSH对绵羊卵母细胞体外成熟具有显著的促进作用,是在减数分裂的恢复后到减数分裂完成之间的某一阶段起的促进作用。     

不同培养时间对家猫卵母细胞核成熟的影响

为了研究不同培养时间对家猫卵母细胞核成熟的影响,以M 199为基础培养液,并添加FSH、LH,分别培养24、28、32、36和43 h后,观察第一极体排出,然后用地衣红染色,将排出第一极体和染色体处于MⅡ期作为卵母细胞成熟的参考判断标志。结果表明,家猫卵母细胞随着培养时间的延长其成熟率也随之增加。成熟培养43 h,极体排出率极显著高于其他各组（P<0.01）,达到66.89%;而24 h组极体排出率为31.51%,极显著低于其他各组（P<0.01）;28、32、36 h各组间差异不显著（P>0.05）。经地衣红染色后,到达MⅡ期的卵母细胞比例与极体排出率趋势相同。43 h组退化率极显著高于其他各组（P<0.01）,达到22.30%。因此,综合考虑成熟率和退化率两项指标,28～36 h为家猫卵母细胞体外核成熟的最佳培养时间。     

不同核移植方法对牛体细胞核移植效率的影响

以牛皮肤成纤维细胞为供体细胞,比较了电融合法和细胞质内注射法2种核移植方法对体细胞核移植效率的影响.电融合法构建重组胚的效率显著低于细胞质内注射法(47.1%比89.0%,P＜0.01);重组胚培养36 h后的裂卵率和培养8 d时囊胚发育率无明显差异(76.4%比73.2%,11.2%比12.3%;P＞0.05),但相对于操作的卵母细胞总数而言,电融合法得到的总囊胚发育率显著低于细胞质内注射法(5.6%比10.9%,P＜0.01).结果表明,用细胞质内注射法进行体细胞核移植效率更高.将发育到桑椹胚和囊胚期的95枚核移植胚胎移植到33头受体牛,其中31头受体牛在移植后第2个情期前返情;1头受体牛在移植后75 d返情,但未进行直肠检查,无法确定妊娠情况;1头受体牛在移植后60 d未见返情,直肠检查确证妊娠,93 d受体牛流产.胚胎移植结果表明,用细胞质内注射法构建的体细胞核移植胚胎至少可以维持早期妊娠.