小鼠卵母细胞体外成熟、体外受精的效果观察

目的　研究不同培养条件对小鼠卵母细胞体外成熟及体外受精率的影响。方法　小鼠卵母细胞分别在含有FSH、BSA和胰岛素的培养液中体外成熟,在Whitten 氏液中体外受精,比较体外成熟率、体外受精率。结果　1- 裸卵(DO) 的体外成熟率、体外受精率(81-4% ,31-0 % ) 均高于卵丘卵母细胞复合体(COC)(48-6 % ,27-1% ) 。2- 在培养液中添加FSH、胰岛素和BSA,卵母细胞的体外成熟率为77-9 % ,82-3% 、60-7% ;体外受精率为77-2 % 、72-6 % 、26-7% ;2 - 细胞率为49-2 % 、34-2 % 、10-0% 。胰岛素组的卵母细胞IVM 率最高,但IVF率、2 - 细胞率低于FSH 组。3- 添加BSA的两组的体外受精率只有26-7 % 、25-8 % ,显著低于其他组,其体外成熟率也较添加FSH 和胰岛素的组成。4- 排出第一极体(PbI) 的卵母细胞的体外受精率和2 - 细胞率(85-9 % ,22-4% ) 均高于GV期卵母细胞(71-1 % ,12-9 % ) 。结论　1- 卵丘卵母细胞(COC) 较裸卵(DO) 的体外成熟率、体外受精率都低,差异显著(P成熟< 0-01;P受精< 0-05) 。2-FSH 和胰岛素均能提高小鼠卵母细胞的体外成熟率、体外受精率。3-BSA可以降低小鼠卵母细胞体外受精率,差异极显著。4-GV 期卵母细胞的体外受精率显著低于体外培养的排出第一极体的卵母细胞(P2 - cell < 0-05,P受精<0-05)     

体外生产胚胎技术

体外生产胚胎技术是胚胎工程技术的基础,其过程涉及到卵母细胞的体外成熟、精子获能和体外受精、胚胎的体外培养这三个方面。主要综述了卵母细胞体外成熟的影响因素、精子分离和获能的方法、早期胚胎体外培养的发育“阻滞”现象和体外培养体系,同时展望了该技术的发展前景 。     

原位PCR技术及其应用

本文介绍了原位PCR的主要进展、基本步骤,并对其在外源基因检测、基因变异、基因表达及定位等方面的应用作一综述 。     

原位PCR和原位杂交检测蛋鸡J亚群禽白血病病毒

根据ALV_J原型株HPRS10 3株gp85基因的内部序列 ,和pol基因的 3′端设计一对引物H5 H7。从发生ML病死蛋用型鸡的肿瘤、骨髓、肝脏、脾脏和输卵管组织中提取总RNA ,反转录为cDNA ,经PCR扩增得到长度为 5 4 5bp的ALV_JcDNA特异性探针。探针定位于 5 2 5 8～ 5 80 2bp。将病鸡的组织石蜡切片置HybaidExpress原位PCR仪平台上 ,以H5 H7为引物进行原位PCR扩增。应用地高辛标记的cDNA探针对原位PCR扩增后切片进行了原位杂交检测。结果在待检组织肿瘤组织、十二指肠、骨髓中出现明显的阳性信号。睾丸、肺、胰腺、大脑、输卵管、肾脏均检出散在的阳性信号。这是国内外首次从分子水平证明蛋鸡J亚群禽白血病。     

植物离体组织染色体加倍诱导同源四倍体

随着生物技术的迅速发展,通过植物离体组织人工诱导多倍体已经成为获得多倍体植株的有效途径。本文就植物离体组织染色体加倍诱导同源四倍体的研究进展做一介绍,详细评述了植物离体组织细胞加倍的途径、影响植物离体组织加倍的因素、利用不同诱导剂进行处理效果比较及离体组织材料的早期倍性鉴定技术等,并展望了植物离体诱导同源四倍体的前景。     

鱼籽兰(Chloranthus errectus)的离体繁殖

本研究通过几种培养基对鱼籽兰外植体培养,选择出了有效的外植体和适合的培养基,并摸索出了有效的移载和栽培技术,培育出了外植体繁殖的成熟开花植株,为该植物提供了一条新的快速繁殖途径,对同属其它植物的快速繁殖具有参考意义。     

四种观赏凤梨的离体繁殖

以粉菠萝[Aechmea fasciata (Lindl.)Baker]、八宝剑（Vriesea poelmanii Hort.)、虎斑姬凤梨[Cryptanthus zonatus(Vis.)Beer]、七彩羞凤梨[Neoregelia carolinae (Beer)L.B.Sm.]等的幼株茎段为外植体,以MS＋NAA 0.5mg L^-1 BA 5mg L^-1为诱导培养基,MS＋BA 1mg L^-1 NAA 0.1mg L^-1为增殖培养基,可获得较好增殖效果。用1／2MS＋NAA 0.5mg L^-1生根效果较好,以椰糠：砂＝1：1,或椰糠：泥炭土：蛭石＝1：1：1为基质移苗的成活率均达95％以上。     

越橘属植物克隆的体外繁殖

以美国引进的越橘属植物为材料,取其成龄植株的茎段或茎尖为外植体,培养在添加各种激素配比的改良WPM的培养基上,研究了影响克隆体外繁殖的因素,筛选离体最佳增殖和生根培养基及培养条件.结果表明:在改良WPM添加Zt 1.0 mg/L的增殖培养基上,增殖系数最大达50,在1/2WPM IBA 0.3 mg/L的生根培养基上生根率因外植体的基因型不同而有差异,可达到30%～70%.通过实验室的半无菌炼苗后,试管苗移栽到温室的成活率达60%.     

原位杂交技术及其在果树研究中的应用

原位杂交技术是近年来快速发展起来的一门新技术,本文介绍了原位杂交技术的基本原理、方法及其发展前景,以及该技术与其它生物学技术相结合而形成的一些新技术。综述了这些技术在果树研究中的应用情况。     

In vitro and in vivo chromosomal aberrations induced by megazol

With the re-emergence of Human African Trypanosomiasis (HAT) on the one hand, which are increasingly resistant to current therapies, and the stage-dependent effectiveness or even the prohibitive cost of these therapies on the other hand, megazol, a 5-nitroimidazole thiadiazole highly active against various trypanosomal species, was assessed for its genotoxic potential. Very little information has become available until now. Two batches of megazol were provided by two different suppliers: Far-Manguinhos, a part of the Fiocruz foundation, under the Brazilian Minister of Health, and Delphia, a French company. These two batches, obtained by different synthetic routes, were studied by means of the in vitro micronucleus assay on L5178Y mouse lymphoma cells, in its microscale version. Both batches of magazol displayed a strong genotoxic activity in this screening assay. A second batch from Delphia was then investigated by use of two tests, i.e. the in vitro metaphase analysis with human lymphocytes and the in vivo micronucleus test in rat bone-marrow. Megazol was shown to be a potent inducer of in vitro and in vivo chromosomal aberrations. Although megazol is a potent trypanocidal agent and is orally bio-available, its toxicity dictates that it should not be developed further for the treatment of HAT and Chagas disease. All development work has therefore been discontinued.     

In vitro synthesis of oat globulin

Thermal denaturation of cytochrome P450 is shown to be a complex process which occurs in two steps. The first (about 50°C) takes place in several stages which can be attributed to denaturation of different regions in the cytochrome P450 with different stability. The second transition (about 90°C) is fully reversible and similar to those described for other hemoproteins.     

活体电穿孔法基因导入技术

活体电穿孔法(invivoelectroporation)可将外源基因有效导入靶组织或器官,导入效率较高,并且可在多种组织器官上应用。近年来活体电穿孔法用于转基因研究的报道不断增多,在基因治疗方面的优势也日趋显著,是一种很好的活体基因导入方法 。     

猪卵母细胞的体外受精及多精受精

对用于猪体外受精(IVF)的研究方法和技术,如传统的液滴IVF、透明带下注射精子受精(SUZI)、卵母细胞质内单精注射受精(ICSI)及细管IVF等进行了简述。与其它动物相比,进行猪卵的体外受精研究,多精受精现象特别明显。大量的研究表明,猪卵的多精受精不但与其品种特性有关,而且与卵母细胞成熟的程度、透明带的异常、受精时获能精子的浓度、输卵管分泌物、受精液蛋白添加成分、NaHCO3浓度、咖啡因、pH值以及温度等因素密切相关。     

In vitro metabolic engineering of hydrogen production at theoretical yield from sucrose

Hydrogen is one of the most important industrial chemicals and will be arguably the best fuel in the future. Hydrogen production from less costly renewable sugars can provide affordable hydrogen, decrease reliance on fossil fuels, and achieve nearly zero net greenhouse gas emissions, but current chemical and biological means suffer from low hydrogen yields and/or severe reaction conditions. An in vitro synthetic enzymatic pathway comprised of 15 enzymes was designed to split water powered by sucrose to hydrogen. Hydrogen and carbon dioxide were spontaneously generated from sucrose or glucose and water mediated by enzyme cocktails containing up to15 enzymes under mild reaction conditions (i.e. 37 °C and atm). In a batch reaction, the hydrogen yield was 23.2 mol of dihydrogen per mole of sucrose, i.e., 96.7% of the theoretical yield (i.e., 12 dihydrogen per hexose). In a fed-batch reaction, increasing substrate concentration led to 3.3-fold enhancement in reaction rate to 9.74 mmol of H₂/L/h. These proof-of-concept results suggest that catabolic water splitting powered by sugars catalyzed by enzyme cocktails could be an appealing green hydrogen production approach.     

In vivo proteome dynamics during early bovine myogenesis

Myogenesis is a complex process of which the underlying mechanisms are conserved between species, including birds and mammals. Despite a good understanding of the stages of myogenesis, many of the mechanisms involved in the regulation of proliferation of the successive myoblast generations, the cellular transitions cell proliferation/alignment of myoblasts/fusion of myoblasts into myotubes/differentiation of myofibres and the control of total myofibre number still remain unknown. An in vivo proteomic analysis of the semitendinosus muscle from Charolais foetuses, at three specific stages of myogenesis (60, 110 and 180 days postconception), was conducted using 2-DE and MS. Expression profiles of more than 170 proteins were revealed and analysed using two way hierarchical clustering and statistical analysis. Our studies identify, for the first time, distinct proteins of varied biological functions and protein clusters with myogenic processes, such as the control of cell cycle activity and apoptosis, the establishment of cellular metabolism and muscle contractile properties and muscle cell reorganisation. These results are of fundamental interest to the field of myogenesis in general, and more specifically to the control of muscle development in meat producing animals.     

In vitro bulblet production of Lachenalia

In vitro bulblet formation and subsequent transplanting of bulblets to soil were studied in order to develop a cost-effective method for the mass production of three Lachenalia varieties. Clumps of adventitious shoots regenerated from leaf explants were used. Bulblet formation was initiated after 2 weeks when shoots were subjected to low temperature (4–15 °C). The size (age) of the adventitious shoot affected the bulblet size, and shoots shorter than 4 mm did not form bulblets. Larger bulblets formed on medium containing 6% sucrose compared to 3% sucrose. Following bulblet initiation, illumination was not necessary for the completion of bulblet formation. Bulblets went into dormancy 3–4 months after they had been initiated or when the culture medium dried out, and they were released from dormancy when the natural night temperatures started to decrease in the late summer. The survival rate of the bulblets after transplanting was directly correlated to the size of the bulblets.The most important factors influencing in vitro bulblet formation of Lachenalia were sucrose concentration, temperature and length of explant shoots. Received: 12 June 1998 / Revision received: 8 September 1998 / Accepted: 23 September 1998     

In vivo mutagenicity studies with iodinated glycerol azeo

Previous studies have shown that iodinated glycerol azeo is positive in a number of in vitro mutagenicity assays including the Ames assay (TA100; TA1535), mouse lymphoma assay, Chinese hamster ovary (cytogenetic) assay and in one in vivo study, the sex-linked-recessive-lethal assay in Drosophila. Prior studies have also shown that the drug is negative in the mouse micronucleus assay. We now report that the drug is also negative for mutagenic activity in a number of other in vivo tests. Single intraperitoneal doses of 25, 125 and 250 mg/kg were without effect in the rat bone marrow chromosomal aberration assay. Single oral doses of 30, 75, 150 and 300 were negative in the rat hepatocyte DNA-repair assay. Single intraperitoneal doses of 30 and 100 mg/kg were without effect in the sister chromatid exchange (SCE) assay in the mouse. Statistically significant effects were seen at 200 and 300 mg/kg in the initial SCE assay and at 300 and 350 mg/kg in the confirmatory SCE assay. The rationale for considering the SCE results to be anomalous and thus not relative to the overall safety evaluation of the drug is presented.     

In vitro and in vivo interactions of aluminum on NTPDase and AChE activities in lymphocytes of rats

Al adjuvants are used in vaccines to increase the immune response. NTPDase and AChE play a pivotal role and act in the regulation of the immune system. The effect of Al exposure in vitro and in vivo on NTPDase and AChE activities in the lymphocytes of rats was determined. In vitro, ATP hydrolysis was decreased by 20.4% and 17.3% and ADP hydrolysis was decreased by 36.5% and 34.8%, in groups D and E, respectively, when compared to the control. AChE activity was increased by 157.3%, 152.5%, 74.7% and 90.8% in groups B, C, D, and E, respectively, when compared to the control. In vivo, ATP hydrolysis was increased by 85% and 86% and ADP hydrolysis was increased by 104.2% and 74%, in Al plus citrate and Al groups, respectively, when compared to the control. AChE activity was increased by 50.7% in Al plus citrate and by 28.6% in Al groups, when compared to the control. Our results show that Al exposure both in vitro and in vivo altered NTPDase and AChE activities in lymphocytes. These results may demonstrate the ability of Al to elicit the immune system, where NTPDase and AChE activities can act as purinergic and cholinergic markers in lymphocytes.     

Drug Transfer Across the Blood-Brain Barrier: Correlation Between In Vitro and In Vivo Models

To assess the drug transport across the blood-brain barrier (BBB), we compared the maximal brain extraction values at time 0 [E(0) values] obtained using either in vitro or in vivo methods. The in vitro BBB model consisted of a coculture of brain capillary endothelial cells growing on one side of a filter and astrocytes on the other. The in vivo model used intracarotid injection in anesthetized rats. Eleven compounds were tested. They were selected because they exhibit quantitatively different brain extraction rates: very low for inulin and sucrose, low for oxicam-related nonsteroidal antiinflammatory drugs and diclofenac, and high for propranolol and diazepam. As these compounds are apparently transferred by a passive diffusion mechanism, two others, glucose and leucine, were added that cross the BBB by a known carrier-mediated process. The in vivo and in vitro E(0) values showed a strong correlation as indicated by the Spearman's correlation coefficient (r = 0.88, p less than 0.01). The relative ease with which such cocultures can be produced in large quantities could facilitate the screening of new centrally acting drugs.