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Techniques in embryo technology (such as in vitro production of embryos and animal cloning) need large quantities of high quality oocytes. But the quality of in vitro matured oocytes from slaughtered animals is generally lower than that of the in vivo matured oocytes. It is usually thought that the reason for this poor quality in in vitro matured oocytes is the lack of capacitation during the dominancy of follicular development in vivo.

目前胚胎工程技术研究和开发(如体外生产胚胎和体细胞克隆等)需要大量高质量的成熟卵母细胞,但利用屠宰动物卵巢卵母细胞经过体外成熟培养而获取的卵母细胞质量还远不如体内成熟卵母细胞,其原因一般认为是由于缺乏体内主卵泡阶段的获能作用。

Calcium sulfate has been used for bone regenation in oral medicine. Osteopontin, a marker molecule, expressed by osteoblast, plays multiple roles in human body. However, the effect of calcium sulfate on osteopontin expressed in osteoblasts is unclear. In this study, U-2 osteosarcoma osteoblastic cells were cultured in 0, 0.5, 1, or 10 μM calcium sulfate hemihydrate. There was no effect in the proliferation of cells, suggesting that calcium sulfate up to 10 μM is biocompatible. The cells and conditioned media were collected for analyzing mRNA and protein levels of osteopontin by reverse-transcription polymerase chain reaction and Western blot, respectively.

硫酸钙用於口腔医学中的骨头再生已经有多年历史,骨桥素是一种被骨母细胞分泌且在人体扮演重要多重角色的标的分子,然而,硫酸钙在骨母细胞分泌硫酸钙上的影响仍未明朗,在本实验之中,培养U-2骨肉瘤类骨母细胞(U-2 osteosarcoma osteoblastic cells)於0、0.5、1、10 μM的硫酸钙之中,在细胞数目的复制上,四种硫酸钙浓度并无影响,显示出U-2骨肉瘤类骨母细胞培养於10 μM的硫酸钙仍是生物相容性的。

Different strains have different reaction to the delay of oviducts in body after animal death. After 10min delay C57BL/6 mouse oocytes have death rate of 56.5%,significantely higher than Kunming mouse (47.6%);Spontaneous activation rates were respectively 13.3% and 46.0%, C57BL/6 were obviously lower than Kunming mouse.4. The oviducts were obtained after being delayed 5min 24h after the mice were injected with hCG. The oocytes were cultured in CZB. About 81.1% occurred spontaneous activation, evidently lower than parthenogenetic rate (96.4%) with SrCl_2. Spontaneous activable oocytes had high cleavage rate(93.2%) and 4-cell rate(87.3%). However, spontaneous activable oocytes had blastula development rate(18.7%) as low as parthenogenetic oocytes by SrCl_2(22.9%).

不同品系小鼠卵母细胞对输卵管在体内滞留产生的反应不同,滞留10min C57BL/6系小鼠卵母细胞死亡率为56.5%,显著高于昆明鼠(47.6%);自发激活率分别为13.3%和46.0%,C57BL/6系小鼠显著低于昆明鼠。4.hCG后24h体内滞留5min卵母细胞在CZB中培养自发激活率为81.1%,显著低于SrCl_2孤雌激活率(96.4%);自发激活的卵母细胞有较高的卵裂率(93.2%)和4-cell比率(87.3%),但囊胚率(18.7%)较低,同卵龄的卵母细胞经SrCl_2孤雌激活囊胚发育率为22.9%,差异不显著。

This result indicates that WT1 gene plays an important role in differentiation and development of fetal kidney and may be the factor that promotes metanephric blastemal cell to differentiate into epithelial cell.

结果显示小胎龄肾组织中WT1蛋白在胚基细胞和幼稚肾小球细胞核表达而大胎龄组肾组织中WT1在肾小管细胞胞浆表达,阳性率分别为57.1%(8/14)和46.2%(6/13),提示WT1基因在胚胎肾分化发育的过程中起着重要作用,WT1蛋白可能是促进后肾胚基细胞向上皮细胞分化的调控因子,其表达在时间上和空间上都受到严格的调控,WT1的表达异常可能导致胚基细胞分化停滞。17例肾母细胞瘤WT1蛋白表达阳性率为41.2%(7/17),阳性部位在胚基型和上皮型肿瘤细胞核,表达部位和阳性率与早期胚胎肾相似,其中间质型肾母细胞瘤均为阴性,胚基型和上皮型肾母细胞瘤阳性率70%(7/10),两组间阳性率有显著差异。

The result of frozen MⅡoocytes using three methods indcated that the self-restraintspear method was better than OPS, and the effection of frozen altogether with self-restraintspear method and centrifugal was the best; the frozen effection was not ideal using fluidspear for 8- cell ovocyte.

用三种冷冻方法(OPS法;自制移液枪头法;移液枪头法+离心法)对MⅡ卵母细胞冷冻的结果表明,自制移液枪头法优于OPS法,其中以移液枪头法+离心组的冷冻效果最好;用移液枪头法对体外发育到8-细胞期卵母细胞的冷冻效果不理想。MⅡ期卵母细胞用OPS法冷冻后卵母细胞IVF和电激活的卵裂率都显著高于移液枪头法(P<0.01),电激活后2-细胞卵裂率比IVF高。

To pronuclear formation rate after oocyte in vitro maturation 18 hours and parthenogenticactivation, oocytes derived from SM is significantly higher than oocytes from PM,oocytes derived from IM is significantly lower that of oocytes detrived from PM and SM.

对于孤雌刺激反应而言,无论是何种类型的卵母细胞成熟18小时后,自发成熟卵母细胞激活后形成原核的比率高于被动成熟的卵母细胞,诱导成熟的卵母细胞的原核形成率显著低于自发和/或被动成熟的卵母细胞

Under the transmission election microscope, we had observed that the state of oocytes arid the cumulus cells around them was tightness, and there were plenty of long and thin microvilli playing the role of connection in the enwrapping. The shape of cumulus cells nucleus was ovate, and the longer and shorter diameters of the cumulus cells were 4.250±0.042μm and 2.750±0.07 μm respectively. The zona pellucida was well proportioned and tightly linking with oocyte. There were great deals of multi-crista mitochondria, lipid droplet and vacuolus. The mitochondria longer and shorter diameters were 0.600±0.106μm and 0.490±0.117μm. Granular nucleoplasma distributed very well in the cell nucleus.

电镜下,卵母细胞外的卵丘细胞紧紧包裹卵母细胞,且与卵母细胞之间有大量细长的微绒毛相联系;卵丘细胞核呈卵圆形,其长径为4250±0.042μm,短径为2.750±0.071μm;透明带均匀,与卵母细胞结合紧密;在皮质区集中分布大量的多峰型线粗体、脂滴和空泡,线拉体长径为0.600±0.106μm,短径为0.490±0.117μm;细胞核中颗粒状的核质分布非常均匀。

In the present study, we collected cumulus cells oocyte complex from ovaries of two different strain mice. The cumulusenclosed oocytes were cultured for 6 h in MEM supplemented with growth factor and FSH. The meiotic maturation of these oocytes has progressed to pro-metaphse Ⅰ stage and the condensed chromosomes are visible under DIC microscope, metaphase Ⅰ spindle even can be detected under Polscope. The metaphase Ⅰ spindles of oocytes were exchanged under such microscopes. After electric stimuli, 91. 6% and 91. 6% karyoplasts-cytoplasm pairs were fused respectively. The resulting oocytes were cultured further in MEM and over 80% of oocytes released the first polar body. 79% and 77% of oocytes formed two pronuclei after in vitro fertilization and the embryos were cultured in KSOM supplemented with amino acids. Over 60% of embryos developed to blastocyst stage.

在本研究中我们在取得两种不同品系小鼠的卵丘卵母细胞复合体后,先将卵丘卵母细胞复合体置于含有多种生长因子和激素的MEM培养液中培养6小时,此时卵母细胞已进入第一次减数分裂的前中期,并且在DIC倒置显微镜下可以看到浓缩的染色体,用Polscope可以发现明显的纺锤体,借助这种显微镜通过显微操作将两种不同品系小鼠来源的卵母细胞的MI纺锤体进行互换,经过三次直流电脉冲作用后,分别有91.6%的胞质—MI核质体对融合,经过进一步的培养后,超过80%的重组卵母细胞排出第一极体,体外受精后分别有79%和77%的重组卵形成双原核,受精后的胚胎在KSOM胚胎培养液中体外培养4天后,超过60%的胚胎发育至囊胚。

However, when two maturing oocytes fused and two spindle will form in the big cell, the chromosomes will not intermingle. In the present study, we removed GV from one oocyte and transfer to the perivetelline space of another GV oocyte. After fusion the resulting oocytes which contained two GVs were cultured further in MEM.

在本实验中,我们利用小鼠GV期卵母细胞将一枚卵母细胞的生发泡取出后移至另一未经去核处理的GV期卵母细胞透明带下,经三次电脉冲作用后将融合的含有两个GV的卵母细胞放入MEM成熟培养液中培养,在培养成熟的不同阶段分别收集卵母细胞进行免疫荧光染色观察微管及核的变化。

The results showed that after 2 h of culture in MEM the chromosomes of oocyte were seperated to form telophase I while a small spindle was observed around chromosomes of primary spermatocyte. However, two clear spindles were observed in the oocytes cultured in CB containing MEM. After further culture, the chromosomes of both primary spermatocyte and oocyte intermingled and formed one large spindle.

在无CB的培养液中培养的卵母细胞培养2小时后,卵母细胞已经进入第一次减数分裂的后期,染色体开始被拉向两极,而精母细胞的MI纺锤体才刚刚形成,虽然继续培养两者染色体可以合二为一并形成一个纺锤体,但是有些染色体发生滞后;当卵母细胞在含有CB的培养液中培养2小时后,在卵母细胞内形成两个相似大小的MI纺锤体,进一步培养形成一个大的纺锤体,染色体正常。

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