新生小鼠卵巢组织的玻璃化冻存、移植及分离卵泡的体外成熟培养初步研究

目的:系统地探索新生小鼠卵巢组织的玻璃化冻存建立卵巢库,移植和分离卵泡以及体外成熟培养的实验方法。方法:对1日龄小鼠卵巢组织进行冻存,解冻复苏和同种肾包膜下移植,从卵巢移植物种中进行卵泡分离和体外成熟培养。结果:①采用平衡液(ES)处理25min,玻璃化液(VS)处理3min方案冻存的卵巢组织具有更高比例的形态完整卵泡,其完整原始卵泡率均值达96.6%,显著性高于实验中其它四组方案(P0.05);②在分别移植2周和4周后回收卵巢移植物,发现二者的卵巢回收率无显著差异(P0.05),但移植4周组的卵泡回收数目要明显高于2周组(P0.05);③在培养基中添加适量的自体血清(10%,V/V)能显著提高卵子的体外成熟率,培养12h后对照组中生发泡破裂(GVBD)发生率为(34.74±4.26)%,添加血清后提高至(54.60±3.37)%,成熟的MⅡ期卵子获得率从(43.17±1.31)%升高至(57.75±5.31)%,有显著性差异(P0.05)。结论:通过该实验较好地建立了卵巢组织的玻璃化冻存、移植和卵泡分离以及体外成熟培养的实验方法。     

微囊化基因工程细胞移植的研究进展

微囊化基因工程细胞移植是将目的基因通过基因转染技术导入到靶细胞内,再将该细胞微囊化后植入受体体内,具有组织相容性好,避免了机体的排斥反应(免疫隔离),且微囊内的功能细胞可以长期存活,发挥其生物学效应。该技术使得异种组织细胞或基因工程细胞移植成为可能,在神经内分泌及代谢疾病等方面的研究取得了可喜的进展,具有广阔的应用前景。     

异体异位卵巢移植对卵巢切除后大鼠生理生化指标的影响

目的探讨异体异位卵巢移植大鼠在生理、生化指标方面的影响变化情况。方法24只健康成年雌性大鼠,随机分为三组,每组8只。Ⅰ组：去势组;Ⅱ组：经新鲜卵巢移植组;Ⅲ组：经冷冻卵巢移植组。观察术前（第0个月）、术后1个月、术后2个月、术后3个月、术后4个月的体温（T）、体重（W）变化情况及甘油三酯（TG）、高密度脂蛋白胆固醇（HDL-C）、低密度脂蛋白胆固醇（LDL-C）、血钙（Ca）、总胆固醇（TC）。     

细胞微囊化免疫隔离技术在移植医学中的应用

作为一种十分有效的免疫隔离技术,细胞微囊化可排除细胞移植中出现的宿主与移植物之间的双向排斥作用,从而使能分泌生物活性物质的细胞在移植后得以存活。目前报道的多种微囊材料中,以海藻酸钠一聚赖氨酸一海藻酸钠的应用最为广泛,可通过提高其生物相容性来减弱免疫排斥反应。细胞微囊化在医学治疗上正在发挥越来越大的作用,特别是基因修饰细胞日益成为研究的焦点。尽管该技术尚需改进,但它在异体和异种组织或细胞移植等方面有着广阔的应用前景。     

新生小鼠卵巢移植雄鼠肾囊下卵泡的生长发育

将1日龄小鼠卵巢移植入成年雄鼠肾囊下,分别于移植后18d、36d回收移植卵巢进行形态学、组织学观察,以评价卵巢移植体在成年雄性受体小鼠体内生长及卵泡发育潜能。结果表明:移植体生长增大,有各级生长卵泡发育;18日龄移植体平均直径为1881.1μm±204.7μm,与1日龄卵巢相比差异极显著(P<0.01),卵泡发育到有腔卵泡阶段;36日龄移植体平均直径达2575.3μm±466.4μm,显著大于18日龄移植体(P<0.01),有成熟卵泡出现,未观察到黄体;从移植体分离到GV期卵母细胞和卵丘卵母细胞复合体。研究表明1日龄小鼠卵巢移植体在雄性受体生理环境中具有正常生长发育和形成成熟卵泡的潜能。     

微囊化干细胞及其应用研究进展

干细胞极强的自我更新能力和多向分化潜能使其可以成为绝佳的种子细胞来源,用于各种疑难疾病的治疗。微胶囊不仅可以为细胞提供三维生长微环境,而且具有良好的免疫隔离性能和生物相容性。微囊化干细胞技术为干细胞大规模、高活性体外培养及长期保存提供了新的技术支持,为细胞移植疗法开辟了新途径。以下首先简述了微囊化技术的发展情况,然后介绍了目前用于微囊化干细胞的材料、制备方法及其免疫隔离作用,重点阐述了近年来微囊化各种不同类型干细胞的研究和应用进展。最后,提出目前微胶囊化干细胞的问题所在并对此技术进行展望。     

脉络丛细胞微囊脑内移植前后的物理及生化性能研究

通过测定脉络丛细胞海藻酸盐微囊在大鼠脑内移植前及移植后的物理及生化性能变化,以探讨其应用于移植治疗神经系统疾病的可行性.用海藻酸盐多聚鸟氨酸微囊包裹猪脉络膜细胞,移植至大鼠黑质-纹状体通道,移植前、移植后4个月及6个月分别测定微囊的大小、形态及细胞的活力、分泌蛋白质及神经营养因子的能力、蛋白质组学的变化.脉络膜细胞微囊在移植前、后大小、细胞活力、蛋白质组学分析、分泌蛋白质及神经营养因子的能力无显著变化.海藻酸盐-多聚鸟氨酸CP微囊能有效地防止脉络膜细胞被受体免疫系统所攻击,使得它们能在大鼠的大脑存活6个月以上并不引起不良作用.     

卵巢冷冻保存和移植的研究进展

卵巢冷冻保存和移植是建立卵子库的一项有效技术,近年来取得了很大进展。本文介绍了卵巢组织冷冻保存和移植的方法及近年来的研究情况。     

去势大鼠新鲜、冷冻卵巢移植后激素代谢变化

目的观察去势大鼠经新鲜、冷冻卵巢移植后体内激素代谢变化情况。方法24只去势大鼠经右下腹皮下移植新鲜卵巢（3 d之内乳鼠卵巢）和经过冻存培养后的卵巢（3 d之内乳鼠卵巢）后,检测移植前、移植后1个月2、个月的三碘甲腺原氨酸（T3）、甲状腺素（T4）、促甲状腺激素（TSH）、睾酮（T）、雌二醇（E2）、孕酮（P）、黄体生成素（LH）及卵泡生成素（FSH）。结果冻存卵巢移植前与新鲜卵巢移植前比较：T值升高,有显著性差异（P〈0.01）;冻存卵巢移植后1个月与新鲜卵巢移植后1个月比较差异无显著性（P〉0.05）;冻存卵巢移植后2个月与新鲜卵巢移植后2个月比较：E2升高明显,差异具有显著性（P〈0.01;新鲜卵巢移植后2个月与移植前比较：T4,T,E2,P,LH均有明显升高（P〈0.01;P〈0.05）;冻存卵巢移植后2个月与移植前比较：E2,P增高明显（P〈0.01;P〈0.05）。结论冷冻卵巢移植较新鲜卵巢移植功能恢复明显。     

微囊化重组CHO细胞制备和培养条件的优化

微囊化重组基因细胞移植治疗肿瘤是一种新兴的肿瘤基因治疗方法,然而由于目前微囊化细胞规模化制备和培养技术还不成熟,阻碍了其在临床治疗中的推广与应用。以重组CHO细胞为模型,考察了不同的微囊制备和培养条件对微囊化细胞生长和内皮抑素表达的影响。实验表明,种子细胞所处的生长阶段和细胞接种密度对微囊化细胞生长和内皮抑素表达的影响较大,对数生长期的细胞进行包囊并且细胞接种密度为1×106~2×106cells/mL微囊时微囊内细胞生长良好、内皮抑素表达量高。微囊制备时间对细胞活性和内皮抑素表达也有较大的影响,制备时间延长对细胞的损伤增大,因此制备时间应控制在5h以内。生物微胶囊在制备过程中会造成细胞损伤,而体外培养是恢复细胞活性的良好方法,在培养过程中微囊接种量为5%时对细胞生长和内皮抑素表达有利。     

乳鼠成骨细胞体外培养

目的建立乳鼠成骨细胞体外培养方法,探讨该方法的可行性和应用价值。方法用出生1~3 d乳鼠颅骨,采用多次胶原酶消化法进行细胞体外培养。倒置显微镜观察细胞形态,对其碱性磷酸酶(ALP)活性及矿化能力进行鉴定,并测定细胞生长曲线。结果原代培养24 h后,大量细胞贴壁生长,细胞呈圆形,48 h后,贴壁细胞呈长梭形、三角形或不规则多边形,并且贴壁细胞伸出2~3个突起,胞质透亮、饱满,7 d后细胞铺满整个平皿底面。经鉴定,培养细胞具有体内成骨细胞的生物学特性。细胞接种后第1与第2个24 h为细胞的潜伏适应期,第3与第7个24 h生长曲线基本为线性曲线,是细胞的对数生长期。结论采用胶原酶消化法分离培养成骨细胞的方法切实可行。     

Mouse Offspring Derived from Fetal Ovaries or Reaggregates Which were Cultured and Transplanted into Adult Females

Primordial germ cells (PGCs) and gonia could be promising novel targets and vehicles for manipulation of the mammalian germ line. To make such manipulation a practical possibility, PGCs or gonia must be allowed to produce gametes and offspring after they were isolated from embryos and manipulated in culture. As the first step to develop such research strategy, we obtained offspring from mouse oogonia which were isolated from embryonic ovaries and cultured as dispersed cells before transplantation into female mice as reaggregates.     

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together within a basement membrane. A finite pool of follicles is laid down during embryonic development, when oocytes in meiotic arrest form a close association with flattened granulosa cells, forming primordial follicles. By or shortly after birth, mammalian ovaries contain their lifetime’s supply of primordial follicles, from which point onwards there is a steady release of follicles into the growing follicular pool.The ovary is particularly amenable to development in vitro, with follicles growing in a highly physiological manner in culture. This work describes the culture of whole neonatal ovaries containing primordial follicles, and the culture of individual ovarian follicles, a method which can support the development of follicles from an immature through to the preovulatory stage, after which their oocytes are able to undergo fertilization in vitro. The work outlined here uses culture systems to determine how the ovary is affected by exposure to external compounds. We also describe a co-culture system, which allows investigation of the interactions that occur between growing follicles and the non-growing pool of primordial follicles.     

Gene Expression Profiling of Cultured Cells From Brainstem of Newborn Spontaneously Hypertensive and Wistar Kyoto Rats

The spontaneously hypertensive rat (SHR) is a good model to study several diseases such as the attention-deficit hyperactivity disorder, cardiopulmonary impairment, nephropathy, as well as hypertension, which is a multifactor disease that possibly involves alterations in gene expression in hypertensive relative to normotensive subjects. In this study, we used high-density oligoarrays to compare gene expression profiles in cultured neurons and glia from brainstem of newborn normotensive Wistar Kyoto (WKY) and SHR rats. We found 376 genes differentially expressed between SHR and WKY brainstem cells that preferentially map to 17 metabolic/signaling pathways. Some of the pathways and regulated genes identified herein are obviously related to cardiovascular regulation; in addition there are several genes differentially expressed in SHR not yet associated to hypertension, which may be attributed to other differences between SHR and WKY strains. This constitute a rich resource for the identification and characterization of novel genes associated to phenotypic differences observed in SHR relative to WKY, including hypertension. In conclusion, this study describes for the first time the gene profiling pattern of brainstem cells from SHR and WKY rats, which opens up new possibilities and strategies of investigation and possible therapeutics to hypertension, as well as for the understanding of the brain contribution to phenotypic differences between SHR and WKY rats.     

未授粉子房和胚珠离体培养诱导植物雌核发育研究进展

未授粉子房和胚珠培养离体诱导雌核发育的研究已近半个世纪,目前共有20科40余种种子植物的未授粉子房或胚珠离体培养诱导了雌核发育,但成功获得雌性单倍体的植物只有10科25种2变种。本文重点对离体雌核发育的胚胎学的研究资料进行综述,包括离体条件下胚珠中胚囊的发育状况、雌核发育的模式、雌性单倍体形成的途径以及自发胚乳的离体诱导并指出该领域研究中存在的问题,进一步在研究方法上提出了设想和建议。     

NMR of Microencapsulated Fish Oil Samples During In Vitro Digestion

The use of nuclear magnetic resonance (NMR) spectroscopy for characterising microencapsulated tuna oil powders (25% and 50% w/w oil) and assessing the behaviour of the microcapsules on their exposure to water, simulated gastric fluid or to sequential exposure of simulated gastric and intestinal fluids was examined. The matrices used for encapsulation were physical mixtures of casein or whey protein in combination with carbohydrates (dextrose monohydrate with either dried glucose syrup or a physically modified resistant starch) or heated mixtures of these matrices. Solid-state ¹³C cross-polarised magic angle spinning NMR and dipolar de-coupled magic angle spinning NMR record the ¹³C NMR signals of the encapsulant material and that of the encapsulated oil, respectively. ¹H and ¹³C solution NMR were used to investigate the relative increase in mobility of the various encapsulant matrices due to their dissolution on exposure to gastrointestinal fluids. The results suggested that the dissolution characteristics of matrices of microencapsulated oil powders were dependent on the type of milk protein and carbohydrate used and whether the protein–carbohydrate matrices were heat-treated prior to encapsulation of the oil.