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We initiated a genetic screen for Drosophila olfactory genes by means of SG18.1-Gal4/UAS system: The result validates our screen as an rapid, effective approach for recovering genes controlling glomerular map patterning; From a misexpression screen of 1,515 P{GS} lines, we identified 23 genes that, when forcibly expressed in the olfactory receptor neurons, disrupted the stereotyped anatomy of the Drosophila antennal lobes; These genes, which have not been shown previously to control olfactory map development, encode novel proteins as well as proteins with known roles in axonal outgrowth and cytoskeletal remodeling.

本文首次利用SG18.1-Gal4/UAS基因筛选系统对果蝇嗅觉基因进行了筛选和鉴定:结果表明,所采用的方法可以快速、有效地分离到果蝇嗅觉相关基因;从1515株果蝇P{GS}品系分离到86株突变体,其中40株有明显表型,从中鉴定了23个基因,这些基因在ORN强迫表达时可以损坏果蝇嗅叶的固有结构;在筛选中发现的一些调节嗅觉图谱发育的基因可以编码新蛋白或编码参与轴突生长和细胞骨架重塑的蛋白;为了便于研究,根据基因的功能和所编码蛋白的特点,我们对分离到的基因进行了分类,即参与轴突引导和突触产生、调节转录、调节细胞骨架和功能未确定或未知的基因;并通过对嗅觉感觉器的检测发现,除了调节转录的基因外,其他基因对触觉器的数量和分布影响不明显。

The results showed that the rates of mature oocyte were 75.2%, 73.1%, 69.8%, 63.5%and oocyte at telophase of MI were 16.3%, 15.9%, 16.9%, 27.0%, respectively. The rates of maturation of oocytes cultured in TCM199 with serum, EGF and TGFαwere significantly higher than that of with BSA team (P<0.05) and the number of oocyte stayed at telophase of MI in TCM199 with serum, EGF and TGFαteam were significantly less than that with BSA team(P<0.05); Significant higher rates (66.6%, 66.6%, 73.6%) of normalα-tubulin distribution in oocytes cultured in TCM199+ serum, EGF and TGFαwere compared to that of TCM199+BSA(43.3%)(P<0.05). The rates of oocyte with cortical granules in cortex were 58.8%, 33.9%, 54.7%and 47.9%respectively, there was significant difference between oocytes cultured with serum, EGF and BSA(P<0.05). In conclusion, TGFαand EGF can promote the oocyte nuclear transition from telophaseⅠto metaphase of meiosisⅡ, and improved the expression and distribution ofα-tubulin during the ovine oocytes maturation; EGF and serum could promote oocyte cytoplasm maturation. The results suggested that EGF and TGFαmight substitute some substance in serum to improve the quality of oocyte nucleus maturation in vitro, but EGF might be more functional than TGFαto promote the maturation of ovine oocyte ooplasm.

三、EGF和TGFα对卵母细胞成熟的影响未成熟卵母细胞分别在TCM199基础培养液+血清、TCM199基础培养液+BSA+EGF、TCM199基础培养液+BSA+TGFα和TCM199基础培养液+BSA四组成熟培养系统中成熟培养,22小时后上述各组卵母细胞的核成熟率分别为75.2%、73.1%、69.8%、63.5%,处于第一次减数分裂末期的比率分别为16.3%、15.9%、16.9%、27.0%,在添加血清、EGF、TGFα各组中核成熟率显著高于其它各组(P<0.05),处于末期的比例明显低于其它各组(P<0.05):添加血清、EGF、TGFα各组在a-Tubulin蛋白的分布与表达上(66.6%、66.6%、73.6%)明显高于BSA组的正常率43.3%(P<0.05);各组CG发生迁移较好的卵母细胞的比率分别为58.8%、33.9%、54.7%、47.9%,血清组和添加EGF组CG迁移至皮质区的比例明显高于仅添加BSA处理组(P<0.05)。

On the termination date, the cultured explants were all examined by Western blot, HE and transmission electron microscope. Our results showed that after 12-days in culture, the cultivation treated with AS-ODN reduced the synthesis of AMBN and had a deformed dental cusp with thinner enamel matrix. Ultrastructure analyses showed that there was hardly any cisternae of the rough endoplasmic reticulum in the ameloblasts at the tip of the cusp of AS-ODN treatedexplants. However, on average the enamel matrix was thinner compared with that in the control group. Furthermore, the collagen fibers in extracellular matrix were found disorganized. These findings seemed to provide a direct experimental evidence that tended to indicate that the arrested AMBN translation in cultured tooth germs might result in the delay of the tooth development.

经用Western蛋白印迹检测表明,所设计的反义核酸对AMBN InRNA具有良好的封闭效果并成功阻断了牙胚对AMBN的表达;在缺乏AMBN情况下,与对照组相比,实验组牙胚在体外可以继续生长发育至钟状晚期,出现成釉细胞和成牙本质细胞的分化,成釉细胞可以分化成为分泌期型成釉细胞,胞浆中缺少合成蛋白质所必需的粗面内质网和高尔基氏体,缺乏溶酶体,表明对蛋白合成和脚的能力降低;实验组牙胚有牙尖形成和基质分泌,但牙尖形态异常,基质形成减少,牙尖周围基质最厚处为O.6卜m,明显薄于对照组的5.spin,基质中胶原纤维粗细不等,排列稀疏, 3 第四军医大学硕士学位论文未见钙化现象,充分证明了AMBN在牙胚发育中参与釉质基质形成和矿化过程,影响胶原纤维和牙本质基质的合成,促进成釉细胞对蛋白质的合成和釉质基质蛋白降解。

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