小鼠2,4,8—细胞胚胎卵裂球体外培养的研究

以ＣＺＢ为基础培养液,培养小鼠２、４、８－细胞胚胎的卵裂球,研究葡萄糖、牛磺酸和胰岛素对１／２卵裂球体外发育的影响及１／４、１／８和２／８卵裂球的体外发育规律。２－细胞胚胎卵裂球在ＣＺＢ中和在添加牛磺酸的ＣＺＢ中培养,其囊胚发育率（分别为９２％、８９％）无显差异（Ｐ＞０．０５）。胰岛素在少量葡萄糖存在的情况下,不影响卵裂球的囊胚发育率;在无葡萄糖时,卵裂球的囊胚发育率（３８％）显降低。牛磺酸在     

蝴蝶兰根段的组织培养

１　植物名称　蝴蝶兰（ＰｈａｌａｅｎｏｐｓｉｓＭｅｌｌｅｒＧｏｌｄ“ＮＦＳ”）。２　材料类别　根段。３　培养条件　（１）愈伤组织的诱导及分化培养基：Ｂ５＋ＮＡＡ１．５ｍｇ·Ｌ－１（单位下同）＋ＫＴ０．２＋ＣＭ１５０ｍｌ·Ｌ－１＋３％蔗糖;（２）原球茎增殖培养基：Ｂ５＋ＧＡ０．０５＋ＣＨ１２０＋３％蔗糖;（３）小苗生长培养基：１／２ＭＳ＋２０％香蕉泥＋２％蔗糖;（４）诱导生根培养基：１／２ＭＳ＋ＩＢＡ０．３＋２％蔗糖。上述培养基均加０．２％活性炭,０．５８％琼脂粉,ｐＨ为５．５;培养基在１２１℃高…     

人工基膜对鼻咽癌上皮细胞株（CNE—2）生长的影响

人工基膜（ＡＢＭ）主要以Ⅰ型胶原的水合性胶原丝网为网架,辅上纤维连结蛋白,Ⅳ型胶原和层粘连蛋白等主要基膜糖蛋白制备而成,具海绵状的形态结构。ＡＢＭ可减少胎牛血清用量１０％,提高细胞生活力和延长细胞传代周期。在２－５％血清浓度的情况下,ＡＢＭ可提高ＣＮＥ－２细胞的生长效率,克隆形成率和克隆生长率而抑制细胞的＾３Ｈ－ＴｄＲＡ掺入。提示在体外研究细胞外基质对细胞的影响时应使用低血清培养液。ＡＢＭ是体外诱     

香石竹的叶片培养及植株再生

１植物名称香石竹（Ｄｉａｎｔｈｕｓｃａｒｙｏｐｈｙ－ｌｌｕｓ）,别名康乃馨。２材料类别无菌苗叶片。３培养条件以ＭＳ为基本培养基。分化培养基附加：（１）６－ＢＡ１．０ｍｐ·Ｌ－１（单位下同）＋ＮＡＡ０．３;（２）６－ＢＡ１．０＋ＮＡＡ０．１;（３）６－ＢＡ１．０＋ＮＡＡ０．０５。增殖培养基附加６－ＢＡ０．５＋ＮＡＡ０．１。分化和增殖培养基均加蔗糖３％、琼脂０．７％,ｐＨ５．８。生根培养基为１／２ＭＳ附加ＮＡＡ０．１,蔗糖２％,琼脂０．６％,ｐＨ５．８。培养温度为（２５±１）℃,光照１２ｈ·ｄ－１,…     

巴拉斯自鹤芋的组织培养和快速繁殖

１植物名称巴拉斯白鹤芋（Ｓｐａｔｈｉｐｈｙｌｌｕｍｐａｌａｓ）。２材料类别小苗茎尖或茎段。３培养条件以ＭＳ为基本培养基：（１）诱导分化与生长培养基为ＭＳ＋ＢＡ２ｍｐ·Ｌ－１（单位下同）＋ＮＡＡ０．２＋３％蔗糖;（２）生根培养基为１／２ＭＳ＋ＩＢＡ０．２＋１．５％蔗糖。以上培养基均加０．７５％琼脂,ｐＨ５．８,培养温度２６～２８℃,每日光照１０～１２ｈ,光照度１５００～２５００ｌｘ。４生长与分化情况剪取顶芽或茎段约０．５～１．０ｃｍ长,用７０％～７５％乙醇消毒３０～６０ｓ,以０．１％ＨｇＣｌ２溶液…     

无牛血清IgG细胞培养基（—GFCS培养基）的制备及其在杂交瘤细胞体外培养中的应用

为了制备不含牛血清IgG的细胞培养基（－GFCS培养基）,并研究其在杂交瘤细胞体外培养中的应用,采用蛋白G亲和层析的方法,将含有血清的细胞培养基中的牛血清IgG去除,以制备无IgG的培养基。使用该培养基体外培养杂交瘤细胞后,监测细胞生长和上清抗体浓度。对培养上清中的IgG类单克隆抗体可以采用蛋白G亲和层析进行纯化。与示去除牛血清IgG的培养基相比,－GFCS培养基培养的杂交瘤细胞的生长状况及上清抗体浓度均无明显变化;从－GFCS培养上清中成功纯化出不被血清IgG污染的IgG类单克隆抗体,本文结果表明,采用－GFCS培养基体外培养分泌IgG类单抗的杂交瘤细胞,可以简化上清抗体的纯化工艺。     

裂叶攀树芋的组织培养

１植物名称裂叶攀树芋（Ｐｈｉｌｏｄｅｎｄｒｏｎｓｅｌｌ－ｏｕｍ）,又名春羽。小天使蔓绿绒。２材料类别嫩茎基部。３培养条件ＭＳ为基本培养基。（１）丛生芽诱导培养基：ＭＳ＋６－ＢＡ３ｍｇ·Ｌ~（－１）（单位下同）＋ＮＡＡ０．１;（２）继代增殖培养基：ＭＳ＋６－ＢＡ２＋ＮＡＡ０．２;（３）生根培养基：ＭＳ＋ＮＡＡ０．５。培养基中蔗糖为３％,琼脂为０．７％,ｐＨ５．８,温度２５～２８℃。每天光照１２ｈ,光照度１０００～１５００ｌＸ。４生长与分化情况４．１无菌材料的获得将外植体用自来水洗净表面,然后用７０…     

甜瓜的组织培养与快速繁殖

１植物名称甜瓜（Ｃｕｃｕｍｉｓｍｅｌｏ）品种“状元”。２材料类别顶芽、侧芽。３培养条件初代培养基：（１）ＭＳ＋６－ＢＡ０．２ｍｇ·Ｌ－１（单位下同）＋ＩＡＡ０．２。增殖培养基：（２）ＭＳ＋６－ＢＡＩ＋ＩＡＡ０．２。生根培养基：（３）Ｍｉｌｌｅｒ＋ＩＡＡ０．２;（４）Ｍｉｌｌｅｒ（无激素）。以上培养基ｐＨ均为５．８～６．０。琼脂浓度在（１）、（２）中为０．７％,在（３）、（４）中为０．６％;糖浓度在（１）、（２）中为３％,在（３）、（４）中为２％。培养温度均为（２５±３）℃,光照度２０００ｌｘ,光…     

白皮松和油松雌配子体愈伤组织的诱导和分化

以白皮松（Ｐｉｎｕｓ ｂｕｎｇｅａｎａ Ｚｕｃｃ．）和油松（Ｐ．ｔａｂｕｌａｅｆｏｒｍ ｉｓ Ｃａｒｒ．）的未成熟胚乳,即雌配子体为外植体进行培养,将雌配子体分别接种在添加不同激素种类和不同浓度配比的改良ＭＳ培养基上诱导愈伤组织。经过２０多天的培养,在含有１—６ ｍ ｇ／Ｌ 萘氧乙酸（ＮＯＡ）和０．５ｍ ｇ／Ｌ６－ＢＡＰ及３％ 蔗糖浓度的培养基上诱导产生了愈伤组织,愈伤组织的诱导频率最高为２５％ 。经细胞学观察证明：愈伤组织细胞确为单倍性的,染色体数目为ｎ＝ １２,正常的体细胞染色体数目为２ｎ＝ ２４,并在含有ＡＢＡ 的原诱导愈伤组织培养基上分化出绿色小芽     

重组人粒细胞集落刺激因子缓释微球的研究

目的：研究固体／油／水法制备重组人粒细胞集落刺激因子缓释微球,为开发其缓释剂型进行初步研究。方法：以聚乳酸．聚羟乙酸共聚物（PLGA）为载体材料：用固体／油／水法和水／油／水法制备载rhG-CSF缓释微球;考察粒径大小、外观、包封率等理化性质;用MieroBCA法考察微球的体外释药特性及影响因素;用SEC-HPLC和MTT比色法初步评价了微球制备工艺过程对rhG-CSF稳定性的影响。结果：两种方法制得的微球形态圆整、分散性良好,包封率均超过80％。固／油／水法制得的微球体外释放在2周内可超过90％,而水／油／水法制得的微球在相同的时间内仅释放30％。对于固／油／水法制备过程,SEC-HPLC法测定蛋白无明显聚集体出现,MTT法测定蛋白活性无明显损失。结论：实验证明了固／油／水法制备的PLGA微球可以实现2周以上的体外缓释。     

Frequency of sex chromosomal mosaicism in bovine embryos and its effects on sexing using a single blastomere by PCR

Assessment of nuclear status is important when a biopsied single blastomere is used for embryo sexing. In this study we investigated the nuclear status of blastomeres derived from 8- to 16-cell stage in vitro fertilised bovine embryos to determine the representativeness of a single blastomere for embryo sexing. In 24 embryos analysed, the agreement in sex determination between a biopsied single blastomere and a matched blastocyst by polymerase chain reaction (PCR) was 83.3%. To clarify the discrepancies, karyotypes of blastomeres in 8- to 16-cell stage bovine embryos were analysed. We applied vinblastine sulfate at various concentrations and for different exposure times for metaphase plate induction in 8- to 16-cell stage bovine embryos. The 1.0 mg/ml vinblastine sulfate treatment for 15 h was selected as the most effective condition for induction of a metaphase plate (> 45%). Among 22 embryos under these conditions, only 8 of 10 that had a normal diploid chromosome complement showed a sex chromosomal composition of XX or XY (36.4%) and 2 diploid embryos showed mosaicism of the opposite sex of XX and XY in blastomeres of the embryo (9.1%). One haploid embryo contained only one X-chromosome (4.5%). Four of another 11 embryos with a mixoploid chromosomal complement contained a haploid blastomere with a wrong sex chromosome (18.2%). In conclusion, assessment of nuclear status of 8- to 16-cell stage bovine embryos revealed that morphologically normal embryos had a considerable proportion of mixoploid blastomeres and sex chromosomal mosaicism; these could be the cause of discrepancies in the sex between biopsied single blastomeres and matched blastocysts by PCR.     

Male chromosomes of sea urchin hybrid andromerogones created with cryopreserved sperm

We developed a method for preparing male chromosomes from sea urchin hybrid andromerogones created with cryopreserved sperm. We obtained hybrid andromerogones by heterospermic insemination of Hemicentrotus pulcherrimus non-nucleate egg fragments produced by centrifuging unfertilized eggs in a stepwise saccharose density gradient. The hybrid andromerogones showed cleavage rates of 1%-93%, cleaved successively into two- and four- blastomeres and developed to early blastulae. The morulae or early blastulae were treated with colchicine (0.1-1.0 mg/ml), dissociated into single blastomeres by pippeting, swollen with 7%-10% sodium citrate for 10 min and fixed with methanol:acetic acid (3:1). The fixed cells were dropped on slides and air-dried. The andromerogones for 5 sperm species showed a half of their respective diploid chromosome numbers without chromosome elimination. This method is applicable for analysis of the haploid male chromosome complement in sea urchin species for which only sperm can be obtained.     

Topology of chromosomes 18 and X in human blastomeres from 3- to 4-day-old embryos.

The positions of chromosomes 18 and X fluorescence in situ hybridization signals were analyzed in blastomeres generated from human in vitro fertilization 3- to 4-day-old embryos after preimplantation screening of aneuploidy of chromosomes 13, 16, 18, 21, 22, X, and Y. Fluorescent signal localization compared with a three-dimensional sphere model of random signal distribution revealed significant differences, providing evidence of peripheral localization of chromosome 18 in aneuploid (p=0.0013) and aneuploid/euploid blastomeres (p=0.0011). No differences were found in localization of chromosome 18 in euploid and in chromosome X in euploid and aneuploid blastomeres.     

Production of bovine tetrapolid embryos by electrofusion and their developmental capability in vitro

Optimal conditions of electrofusion for blastomeres of two-cell bovine embryos to produce tetraploid embryos were investigated. The high fusion rate (73–95%), viability, and develop mental capacity were obtained under a field strength of 1.0 kV/cm with direct current pulses of 10 or 25 μsec duration applied twice. Cytological study showed that 78.6% (11/14 embryos) of embryos exposed to electrofusion had tetraploid chromosome sets and the others were diploid or hexaploid. The tetraploid embryos had the capability to develop up to morulae stage in vitro.     

Intravital selection of early mouse embryos by sex and karyotypic characteristics

The possibility of live karyotyping by a single blastomere isolated at the 2-, 4-, and 8-cell stage has been investigated. It is expedient to use to this end a single blastomere isolated from a 4-cell embryo. Three rest blastomeres formed normal morulae or blastocysts upon cultivation during 44 or 68 hrs. When the isolated blastomeres were cultivated for 14-16 hrs in a medium 0.5 micrograms/ml colcemide, 97% of blastomeres were at the metaphase stage and 72% of chromosome plates were suitable for karyotyping. The prospects of the method proposed in experimental embryology and for selection of the early embryos of farm animals by sex are discussed.     

Chromosome analysis of blastomeres from human embryos by using comparative genomic hybridization

Karyotypic studies of aborted fetuses have been used to draw the inference that the proportion of conceptuses with chromosome abnormalities is very high. Fluorescent in situ hybridization (FISH) studies of blastomeres from early cleavage embryos have provided some support for this inference but they are limited to the study of a few chromosomes. We describe the novel application of comparative genomic hybridization (CGH) to the study of numerical and structural abnormalities of single blastomeres from disaggregated 3-day-old human embryos. CGH results were obtained for 63 blastomeres from 12 embryos. Identification of all chromosomes with the exception of chromosomes 17, 19, 20 and 22 was possible. The embryos divided into four groups: (1) embryos with a normal CGH karyotype seen in all blastomeres; (2) embryos with consistent aneuploidy suggesting meiotic non-disjunction had occurred; (3) embryos that were mosaic generally with one or more cells showing aneuploidy for one or two chromosomes but some with cells showing extensive aneuploidy; and (4) one embryo with extensive aneuploidy in all blastomeres. The extensive aneuploidy in group 4 is interpreted as corresponding to the random aneuploidy seen in "chaotic" embryos reported by using interphase FISH. Partial chromosome loss and gain following chromosome breakage was observed in one embryo. Our analysis provides basic biological information on the occurrence of constitutional and post-zygotic chromosome abnormalities in early human embryos. Used in conjunction with embryo biopsy, diagnostic CGH should allow the exclusion of a proportion of embryos that appear normal but that have a poor probability of survival and, therefore, may improve the implantation rate after in vitro fertilization.     

In vitro separation and development of sheep blastomeres

Four cell embryos collected by laparatomy from Sardinian breed ewes superovulated with FSH-p (16 mg Sigma), were divested of their zonae pellucidae (ZP) by micromanipulation or chemical methods (pronase 0.5%, tyrode pH 2.2). The blastomeres were separated by pipetting using a flame polished pasteur pipette in a Ca free medium (PBS. Sigma) and were inserted into previously evacuated Z.P. using a Leitz micromanipulator. The Z.P. were removed either mechanically or with acid tyrode; pronase was unable to digest them after incubation at 30 degrees C for 120 minutes. The single blastomeres were cocultured on a monolayer of ovine oviductal epithelial cells in TCM 199 + 10 FCS at 38 degrees C in 5% CO2 for 60 hours. No developments were observed in blastomeres obtained by acid digestion of the ZP while 50% of the other blastomeres continued their development until the 16 cell stages. Our results suggest that coculture with oviductal epithelial cell monolayers can support in vitro development of single ovine blastomeres.     

Chromosomal diagnosis in each individual blastomere of 5- to 10-cell bovine embryos derived from in vitro fertilization

Chromosomal normality and sex were diagnosed in each blastomere of bovine embryos derived from in vitro fertilization (IVF). Bovine embryos developing to the 5- to 10-cell stage were separated into individual blastomeres with 0.5% protease. After treatment with 100 ng/mL vinblastine sulfate for 8 to 10 h, they were prepared for chromosome samples. In total, 33 bovine embryos and 185 blastomeres were examined. Chromosomal normality was analyzed in 43.8% (81/185) of the blastomeres and 60.6% (20/33) of the embryos; while chromosomal anomalies were found in 16 (80%, 16/20) of the embryos, 5 haploid embryos and 11 mosaic (n/2n) embryos. Mosaicism characteristic of the opposite sex in X-and Y-chromosomes was found in 2 haploid embryos, and that of a Y-chromosome and of XX chromosomes in 1 n/2n embryo. Various sex-chromosome compositions were also observed in the other 10 chromosomal mosaic n/2n embryos.     

Chromosome abnormalities in human arrested preimplantation embryos: a multiple-probe FISH study.

Numerical chromosome abnormalities were studied in single blastomeres from arrested or otherwise morphologically abnormal human preimplantation embryos. A 6-h FISH procedure with fluorochrome-labeled DNA probes was developed to determine numerical abnormalities of chromosomes X, Y, and 18. The three chromosomes were stained and detected simultaneously in 571 blastomeres from 131 embryos. Successful analysis including biopsy, fixation, and FISH analysis was achieved in 86.5% of all blastomeres. The procedure described here offers a reliable alternative to sexing of embryos by PCR and allows simultaneous ploidy assessment. For the three chromosomes tested, numerical aberrations were found in 56.5% of the embryos. Most abnormal embryos were polyploid or mosaics, and 6.1% were aneuploid for gonosomes or chromosome 18. Extrapolation of these results to all human chromosomes suggests that the majority of abnormally developing and arrested human embryos carry numerical chromosome abnormalities.     

小鼠2—细胞胚胎单个卵裂球体外培养及其发育形成囊胚的玻璃化冷冻保存技术的研究（简报）

The present experiments were designed to study the effects of glucose, EDTA, glutamine on the in vitro development of single blastomeres from 2-cell embryos in mouse, and the efficiency of cryopreservation of blastocysts from single blastomers with different vitrification. Single blastomeres derived from female ICR x male BDF1 2-cell embryos were cultured in mKRB with or without glucose, EDTA and glutamine, respectively. The expanded blastocyst rates were significantly different between in mKRB with glucose and without glucose (34% vs 65%); The blastomeres were cultured in mKRB with EDTA and glutamine but glucose, the expanded blastocyst rate (90%) was significantly higher than other groups. The blastocysts derived from single blastomeres were vitrified in liquid nitrogen after equilibration in GFS40 for 0.5-2 min, the survival rate 24%-51%. The blastocysts were pretreated in mPBS with 10% glycerol for 5 min, followed by exposure to GFS40 at 25 degrees C for 0.5 min, then vitrified in liquid nitrogen(two-step method), the survival rate was 61%. However, the survival rates increased to 64% and 70% when the blastocysts were vitrified(one-step method) ater equilibration in EFS40 at 25 degrees C for 0.5-1 min.