植物学教学中的卵菌分类地位

植物学教学中的卵菌分类地位钟恒（中山大学生物系,广州５１０２７５）ＴＨＥＴＡＸＯＮＯＭＩＣＳＴＡＴＵＳＯＦＯＯＭＹＣＥＴＥＳＩＮＴＨＥＢＯＴＡＮＩＣＡＬＴＥＡＣＨＩＮＧＺｈｏｎｇＨｅｎｇ（ＤｅｐａｒｔｍｅｎｔｏｆＢｉｏｌｏｇｙ,ＺｈｏｎｇｓｈａｎＵｎ...     

层粘连蛋白活性肽段对小鼠胚泡着床的影响

层粘连蛋白活性肽段对小鼠胚泡着床的影响ＥＦＦＥＣＴＳＯＦＬＡＭＩＮＩＮＰＥＰＴＩＤＥＳＯＮＢＬＡＳＴＯＣＹＳＴＩＭＰＬＡＮＴＡＴＩＯＮＩＮＭＩＣＥ关键词胚泡子宫着床层粘连蛋白活性肽段ＫｅｙｗｏｒｄｓＢｌａｓｔｏｃｙｓｔ,Ｕｔｅｒｕｓ,Ｉｍｐｌａｎｔａ...     

癌基因EJ－ras在小鼠胚胎干细胞（ES－5）中的表达及其对ES－5细胞一些特征的影响

转基因动物在研究基因的功能、医药、畜牧业等方面有重要的应用。利用胚胎干细胞（ＥＳ）制作转基因动物,比传统的显微注射法有无可比拟的优点。但是,目前、只在小鼠中建立起了ＥＳ细胞系,在其它哺乳动物（包括家畜）中尚未建成。近来,人们开始探索：改变胚胎细胞的遗传物质（如：把外源基因导入胚胎细胞）是否有利于获得ＥＳ细胞系．在各种外源基因中,癌基因是值得尝试的基因。因为：（１）ＥＳ细胞的建系是使胚胎的ＩＣＭ细胞永生化的过程;（２）在体外利用癌基因转染可使许多原代培养细胞永生化;（３）ＥＳ细胞高表达一些原癌基因。我们用癌基因ＥＪ－ｒａｓ转染小鼠ＥＳ细胞,观察其对ＥＳ－５细胞一些特性（增殖、嵌合能力）的影响,以便选择合适的癌基因用于家畜ＥＳ细胞的建系。ＥＳ－５细胞通常培养在丝裂霉素处理的小鼠胚胎成纤维细胞（ＭＥＦ）上（Ｆｉｇ．Ａ）。转染用ＥＳ－５细胞培养在无饲养层细胞的、含有大鼠肝细胞（ＢＲＬ）的条件培养液ＤＭＥＭ中。ＥＪ－ｒａｓ基因克隆在真核表达载体ＰＳＶ２ｎｅｏ的ＢａｍＨＩ位点上、使用磷酸钙法转染ＥＳ－５细胞,Ｇ－４１８法筛选阳性克隆,得到的抗性克隆仍以集落形式生长（Ｆｉｇ．Ｂ）,选取５个较好的克隆（ＥＳ－ｒａｓ１,２,     

小鼠孤雌胚胎干细胞集落的建立

ＥＳＴＡＢＬＩＳＨＭＥＮＴＯＦＳＴＥＭＣＥＬＬＣＯＬＯＮＩＥＳＦＲＯＭＰＡＲＴＨＥＮＯＧＥＮＥＴＩＣＡＬＬＹＤＥＲＩＶＥＤＢＬＡＳＴＯＣＹＳＴＳＯＦＭＯＵＳＥ小鼠孤雌胚胎干细胞集落的建立ＫｅｙｗｏｒｄｓＭｏｕｓｅ,Ｐａｒｔｈｅｎｏｇｅｎｅｔｉｃｅｍ...     

昆明白小鼠1细胞胚胎体外培养系统的研究

研究发现在有或者没有磷酸盐的条件下,葡萄糖均抑制昆明白小鼠ｌ－４细胞期胚胎的体外发 育。在不含葡萄糖和磷酸盐的ＨＥＣＭ－ｌ中,桑椹率为４０．０５％（７４／１６８）,而对照Ｇ－ＨＥＣＭ－１仅为 ８．１４％（７／８６）;不含葡萄糖含有磷酸盐的ＣＺＢ中,桑椹率为６７．１１％（９３／１５２）,而对照ＴＡＬＰ仅 ６· ６７％（６／９０）。用不含葡萄糖而含有１． ０ｍｍｏ１／Ｌ谷氨酸肢和０． １１ｍｍｏｌ／Ｌ ＥＤＴＡ的ＣＺＢ液,与兔输 卵管上皮单层培养细胞（ＲＯＥＣ）协同培养小鼠１细胞胚,７３．３３％（１１０／１５０）胚胎发育至桑椹胚, 但没有观察到囊胚形成、用上述ＣＺＨＲＯＥＣ系统培养小鼠１细胞胚４８小时（３－４细胞）,再移入 ＴＣＭ１９９＋１０％ＦＣＳ＋ＲＯＥＣ系统,有７６．７４％（６７／８６）胚胎发育至桑椹胚,９６／小时后,４０．７０％ （３５／８６）发育至囊胚。     

柑桔种间体配融合及培养研究

“平户”文旦（柚）（Ｃｉｔｒｕｓｇｒａｎｄｉｓ）Ｏｓｂｅｃｋ的四分体经酶解,分离出原生质体。ＰＥＧ（聚乙二醇）诱导这类原生质体与二倍体“伏令夏”甜橙（Ｃ．ｓｉｎｅｎｓｉｓ）胚性悬浮细胞系的原生质体融合。融合后的原生质体培养于ＢＨ３／ＥＭＥ培养基中。２天后,观察到花粉管生长现象。不同处理的结果显示,这一现象来源于异核体细胞。这种具花粉管生长的细胞可进一步分裂,形成多细胞团及球形和心形胚状体。对再生的胚状体进行染色体数检查,证明１３．１％的胚状体为三倍体,２ｎ＝３ｘ＝２７。而起始悬浮细胞系为二倍体,检查的３９２个细胞,未发现有染色体倍性变异。     

青海南部太白韭4居群的核型研究

研究了葱属太白韭青海４个居群的染色体数目和核型。结果如下,居群１：２ｎ＝２ｘ＝１６＝１２ｍ＋２ｓｍ＋２ｓｔ（２ＳＡＴ）,居群２：２ｎ＝２ｘ＝１６＝１４ｍ＋２ｓｔ（２ＳＡＴ）;居群３：２ｎ＝４ｘ＝３２＝２４ｍ＋４ｓｍ＋４ｓｔ（４ＳＡＴ）,居群４：２ｎ＝２ｘ＝１６＝１４ｍ＋２ｓｔ（２ＳＡＴ）＋Ｂｓ（０－２）。并讨论了多倍体和Ｂ染色体形成与分析。     

辽宁地区产6种乌头的细胞分类学研究

对我国辽宁地区毛莨科乌头属６个种的染色体的数目和形态进行了研究,并进行了核型分析。其染色体基数为Ｘ＝８,核型公式为：两色乌头：２ｎ＝２ｘ＝２ｍ＋１０ｓｍ＋４ｓｔ;蛇岛乌头为：２ｎ＝４ｘ＝１０ｍ＋２０ｓｍ（ＳＡＴ）＋２ｓｔ＋２Ｂ;黄花乌头为：２ｎ＝４ｘ＝４ｍ＋１２ｓｍ（ＳＡＴ）＋８ｓｔ＋１Ｂ;北乌头三倍体为：２ｎ＝３ｘ＝２Ｍ＋４ｍ＋１８ｓｍ;北乌头４倍体为２ｎ＝４ｘ＝４ｍ＋２８ｓｍ。同时,对乌头属下     

家兔胚泡ICM在体外培养系统中的行为

一、用显微外科方法从兔早期胚泡分离来的ＩＣＭ在所用的体外培养系统中的行为,因ＩＣＭ的生长状态而不同。呈带状生长的ＩＣＭ从近端向远处延伸,而细胞的分化则从远端逐渐向近端进行,它具有明显的极性。细胞的分化秩序井然,层次分明。因此呈状生长的ＩＣＭ适艇于进行细胞分化和细胞谱系的研究。二、呈团块状生长的ＩＣＭ,没有明显的极性,细胞分化开始于团块的外表面,逐渐向中央进行,分化开始稍晚一些,速度也较慢,这种生长     

小鼠早期胚胎发育期间TGF-β_1免疫组织化学定位

用兔抗人血小板TGF-β_1 N末端1—29氨基酸残基人工合成多肽抗血清作探针以及免疫荧光和免疫酶染色技术,分析了1—12天小鼠早期发育期间胚胎的TGF-β_1物质分布。结果表明,着床前胚胎包括卵裂细胞,桑椹胚和胚泡的ICM及滋养外胚层等细胞均显示TGF-β_1阳性免疫荧光染色。免疫酶染色还证明,沿囊胚腔顶部单层排列的原始内胚层细胞比邻近的ICM细胞有较深的染色反应。随着胚胎着床和进一步发育,7天龄胚胎中胚层早期形成阶段,紧靠中胚层一侧的外胚层胞质中含有浓集的棕色颗粒;各胚层的部分区域也存在着染色强度上差别。8—12天龄胚胎中,体节,心壁、间质细胞和肠道以及卵黄囊的脏壁中胚层均有显著的TGF-β_1免疫酶阳性物质。这些结果表明,着床前小鼠胚胎富含TGF-β_1物质,着床后的胚外组织,例如卵黄囊也为胚胎进一步发育提供了富含TGF-β_1物质的微环境;同时也提示,小鼠早期胚胎发育期间的胚泡形成,ICM细胞分化出原始内胚层,卵柱期中胚层形成,以及以后的神经管、体节和肢芽形成阶段等一系列形态发生和器官形成过程中,TGF-β_1可能是参与重要作用的一种生长调节因子。     

Isolated mouse inner cell mass is unable to reconstruct trophectoderm

The ability of ICM to differentiate into TE is still a controversial issue. Many of authors have showed the reconstruction of TE from isolated ICMs. We showed that immunosurgical method is not 100% efficient and that the original TE cells very often remain on the surface of isolated ICMs. We also found that isolated ICM cells cultured in vitro do not express Cdx2, and that the TE is reconstituted from TE cells which have survived immunosurgery. This indicates that very soon after the formation of TE in the blastocyst, the cells of ICM lose the potency to differentiate into trophectoderm.     

A mRNA landscape of bovine embryos after standard and MAPK-inhibited culture conditions: a comparative analysis

Background

Genes and signalling pathways involved in pluripotency have been studied extensively in mouse and human pre-implantation embryos and embryonic stem (ES) cells. The unsuccessful attempts to generate ES cell lines from other species including cattle suggests that other genes and pathways are involved in maintaining pluripotency in these species. To investigate which genes are involved in bovine pluripotency, expression profiles were generated from morula, blastocyst, trophectoderm and inner cell mass (ICM) samples using microarray analysis. As MAPK inhibition can increase the NANOG/GATA6 ratio in the inner cell mass, additionally blastocysts were cultured in the presence of a MAPK inhibitor and changes in gene expression in the inner cell mass were analysed.

Results

Between morula and blastocyst 3,774 genes were differentially expressed and the largest differences were found in blastocyst up-regulated genes. Gene ontology (GO) analysis shows lipid metabolic process as the term most enriched with genes expressed at higher levels in blastocysts. Genes with higher expression levels in morulae were enriched in the RNA processing GO term. Of the 497 differentially expressed genes comparing ICM and TE, the expression of NANOG, SOX2 and POU5F1 was increased in the ICM confirming their evolutionary preserved role in pluripotency. Several genes implicated to be involved in differentiation or fate determination were also expressed at higher levels in the ICM. Genes expressed at higher levels in the ICM were enriched in the RNA splicing and regulation of gene expression GO term. Although NANOG expression was elevated upon MAPK inhibition, SOX2 and POU5F1 expression showed little increase. Expression of other genes in the MAPK pathway including DUSP4 and SPRY4, or influenced by MAPK inhibition such as IFNT, was down-regulated.

Conclusion

The data obtained from the microarray studies provide further insight in gene expression during bovine embryonic development. They show an expression profile in pluripotent cells that indicates a pluripotent, epiblast-like state. The inability to culture ICM cells as stem cells in the presence of an inhibitor of MAPK activity together with the reported data indicates that MAPK inhibition alone is not sufficient to maintain a pluripotent character in bovine cells.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1448-x) contains supplementary material, which is available to authorized users.     

家兔囊胚在饲养层上的生长行为

研究了家兔囊胚在饲养层上的生长行为。结果表明,滋养层细胞铺展后,形成一层透明的膜状结构。覆盖在内细胞团之上。内细胞团在４８小时后,可见到一定程度的增殖,第３天增殖速度加快。生长的内细胞团可呈团状、条状、网状和混合型四种形态。混合型内细胞团分化出现较早,呈团状、条状和网状生长的内细胞团分化较晚。团状内细胞团一般在原位生长,而呈条状和网状生长的内细胞团增殖很快并迅速扩展到各处。滋养层细胞覆盖于未分化的     

层粘连蛋白及其肽段对小鼠胚泡粘附和扩展的作用

作为细胞外基质的主要成分之一的层粘连蛋白（ＬＮ）,对小鼠胚泡的粘附、扩展有显著促进作用。ＬＮ分子上的一些活性位点对胚泡的粘附和扩展也具有一定的作用,含ＲＧＤ位点序列的合成肽段ＲＧＤＳ对胚泡的粘附有促进作用;含ＹＩＧＳＲ位点序列的合成肽段ｃＹＩＧＳＲ对胚泡的粘附和扩展均有促进作用;且ＲＧＤＳ和ｃＹＩＧＳＲ可以竞争性抑制ＬＮ对胚泡粘附和扩展的促进作用。以上结果表明ＬＮ对胚泡的作用是通过胚泡上不同的ＬＮ     

前列腺素F（PGF）抗血清对小鼠胚泡着床的影响

本文试图利用自制的PGP抗血清,对小鼠子宫局部进行注射,以观察其对胚泡着床的影响。结果表明,于妊娠第3天(孕卵在输卵管阶段)单侧子宫角注射PGF抗血清,对胚泡着床无影响。而妊娠第4天(胚泡在子宫阶段〕单侧或双侧子宫角注射PGF抗血清,对胚泡着床均有明显的抑制作用。这一结果提示小鼠胚泡着床中PGF起着重要的作用。     

人类囊胚期胚胎培养-移植的研究现状

传统的体外受精-胚胎移植选择卵裂期胚胎进行移植,然而,多胎率高、成功率低始终困惑着生殖医学者,囊胚移植更符合生理过程,胚胎与子宫内膜更为同步性,因而可获得更高的胚胎种植率及临床妊娠率,且减少胚胎的移植数量,降低了多胎妊娠发生率,具有广泛的应用前景。     

纤粘连蛋白对小鼠胚胎体外发育和体外着床的作用

应用小鼠胚泡和外胎盘锥体外培养的方法,研究了纤粘连蛋白对小鼠胚泡发育及胚泡或外胎盘锥粘附和扩展的影响。结果显示,纤粘连蛋白对小鼠胚泡发育有一定的促进作用;对胚泡及外胎盘锥的粘附和胚泡初生滋养层细胞及外胎盘锥次生滋养层细胞扩展均有显著促进作用。纤粘连蛋白分子活性位点的合成肽段精－苷－天冬－丝氨酸可有效抑制纤粘连蛋白对胚泡或外胎盘锥发育、粘附和扩展的促进作用。结果表明,纤粘连蛋白在小鼠胚胎发育和着床过     

不同温度条件下小鼠囊胚OPS法玻璃化冷冻保存技术的研究

本实验采用OPS法在不同温度条件下对小鼠囊胚实施冷冻保存,研究用EDFS和EFS溶液冷冻保存囊胚的效率和提供不同温度下筛选玻璃化溶液的依据,为家畜和人类胚胎的冷冻保存建立模型。25℃室温和37℃恒温台条件下OPS一步法冷冻保存小鼠囊胚,EFS40和EDFS40冷冻组扩张囊胚率(92.31%,92.30%)与对照(97.26%)均无显著差异(P>0.05),但EDFS40孵化囊胚率(59.62%)显著低于对照组(83.56%)(P<0.05);二步法冷冻结果显示,采用EDFS30和EFS40均能高效保存小鼠囊胚,解冻后扩张囊胚率(95.69%和95.05%)和孵化率(80.48%和78.95%)与对照无显著差异(P>0.05)。当改为25℃室温不使用恒温台条件下,一步法冷冻的胚胎解冻后,仅EDFS40冷冻组扩张囊胚率和孵化囊胚率(85.96%和75.44%)与对照(96.05%和82.89%)无显著性差异(P>0.05);实施二步法冷冻的胚胎,解冻后EDFS30,EDFS40和EFS40组均获得理想效果,扩张囊胚率(92.03%-95.31%)及孵化囊胚率(67.19%-76.76%)与对照均无显著差异(96.05%和82.89%)(P>0.05)。据体外发育结果,选择最佳冷冻组胚胎移植给假孕4d的受体母鼠,其妊娠率和产仔率(90.90%和37.33%)与新鲜胚对照组(91.67%和42.33%)无显著差异(P>0.05)。结果证实,EDFS30、EDFS40和EFS40三种冷冻液在不同的温度条件和采用不同冷冻程序,均能成功保存小鼠囊胚。     

VITRIFICATION OF IN VIVO AND IN VITRO PRODUCED OVINE BLASTOCYSTS

Although cryopreservation of bovine embryo has made great progress in recent years, little achievement was obtained in ovine embryo freezing, especially in vitro produced embryos. However, a simple and efficient method for cryopreservation of sheep embryos will be important for application of ovine embryonic techniques such as in vitro fertilization, transgenic, cloning and etc. In this study ovine blastocysts, produced in vivo or in vitro, were cryopreserved by vitrification in EFS40 (40% ethylene glycol (EG), 18% ficoll and 0.5 M sucrose) or GFS40 (40% glycerol (GL), 18% ficoll and 0.5 Mol sucrose). In Vitro produced, early blastocysts were directly plunged into liquid nitrogen (LN₂) after preparation by one of the following procedures at 25°C: (A) equilibration in EFS40 for 1 min; (B) equilibration in EFS40 for 2 min; (C) equilibration in EFS40 for 30 s following pretreatment in 10% EG for 5 min; (D) equilibration in EFS40 for 30s following pretreatment in EFS20 for 2 min (E) equilibration in GFS30 for 30 s following pretreatment in 10% GL for 5 min. The survival rates observed after thawing and in vitro culture for 12 h were A 78.0% (39/50), B 50.0% (26/52), C 93.3% (70/75), D 92.0% (46/50) and E 68.0% (34/50). Survival rates were not significantly different for treatments C and D (p>0.05), but those for groups C and D were significantly higher than for A, B and E (p<0.05). After 24 h in vitro culture, hatched blastocyst rates were A 28.0% (14/50), B 21.1% (11/52), C 49.3% (37/75), D 48.0% (24/50), E 32.0% (16/50) and control 54.0% (27/50). The hatching rates for groups A, B and E were significantly lower than the control (p<0.05) in which early IVF blastocysts were cultured in fresh SOFaaBSA medium following treatment in PBS containing 0.3% BSA for 30 min, but for groups C and D it was similar to the control (p>0.05). The freezing procedures A, B and C were used to vitrify in vivo produced, early blastocysts recovered from superovulated ewes. The survival rates of frozen-thawed in vivo embryos were A 94.7% (72/76), B 75.0% (45/60) and C 96.4% (54/56) and for group B was significantly lower than for the other two treatment groups (p<0.05). Hatched blastocyst rates were A 46.0% (35/76), B 26.6% (16/60), C 51.8% (29/56) and the control 56.7% (34/60) in which early blastocysts from superovulation were cultured in fresh SOFaaBSA medium following treatment in PBS containing 0.3% BSA for 30 min. The hatching rate for treatment B was significantly lower than for the control (p<0.05) but did not differ between groups A, C and the control (p>0.05). Frozen-thawed embryos vitrified by procedure C were transferred into synchronous recipient ewes. Pregnancy and lambing rates were similar for embryos transferred fresh or frozen/thawed for both in vivo and in vitro produced embryos. These rates did not differ between in vivo and in vitro embryos transferred fresh (p>0.05). However, for frozen-thawed embryos, both rates were significantly lower for in vitro than for in vivo produced embryos (p<0.05).     

降钙素对胚胎着床的影响机理

降钙素(calcitonin,CT)是甲状腺滤泡旁细胞分泌的一种含有32个氨基酸残基的肽类激素,是动物体内重要的调节钙磷代谢的内分泌因子。近年来的研究发现CT在胚胎着床过程中起着重要的作用。胚胎着床涉及到母体子宫和胚胎之间的复杂而精确的调控。在孕激素作用下,围着床期子宫内膜表达CT,CT与其膜受体结合后可激活腺苷酸环化酶(adenylate cyclase,AC)和磷脂酶(Cphospholipase C,PLC)等激酶的活性,促进细胞外Ca2 内流,从而促使子宫内膜和胚胎发生一系列的变化,有利于胚胎的植入。