鼻咽癌上皮细胞株HNE1差异表达基因的分离与鉴定

为了分离鼻咽癌差异表达基因 ,应用抑制性扣除杂交技术 ,在正向抑制性扣除杂交中 ,以鼻咽癌上皮细胞株HNE1cDNA作为检测子 ,以人胚鼻咽上皮细胞cDNA作为驱赶子 ;在反向抑制性扣除杂交中 ,以人胚鼻咽上皮细胞cDNA作为检测子 ,以鼻咽癌上皮细胞株HNE1cDNA作为驱赶子 ,分别通过抑制性扣除杂交 ,构建了鼻咽癌上皮细胞株HNE1表达下调和表达上调的两个扣除cDNA文库 .从鼻咽癌相关的扣除cDNA文库中随机挑取 1 2 0 0个克隆 ,采用菌落PCR扩增其插入cDNA片段 ,自动点膜制备成cDNA微阵列膜 ,分别用鼻咽癌上皮细胞株HNE1、人胚鼻咽上皮mRNA经逆转录标记cDNA探针 ,分别与cDNA微阵列膜杂交 ,通过杂交信号的自动扫描分析 ,对杂交信号存在 5倍差异的克隆进行测序 ,获得了 1 0个鼻咽癌差异表达基因的cDNA片段 ,其中 3个为新基因序列 ,其GenBank登录号为 :AF5 1 0 1 88、AF5 1 0 1 89和AF5 1 0 1 90 ,7个代表已知基因序列 .采用RT PCR证实S1 0 0A8,CK1 9和RBP1基因在人胚鼻咽上皮中高表达而在鼻咽癌细胞株HNE1中低表达 .这些结果显示上述基因可能是鼻咽癌发生的重要因素     

利用抑制性扣除杂交技术克隆水稻磷饥饿诱导基因

磷素是植物生长所必需的重要元素.在缺磷环境中,植物能够调节自身的形态、生理生化和基因表达水平来适应环境的变化.为研究水稻(Oryza sativa L.)耐低磷胁迫的分子机理,采用抑制性扣除杂交技术(SSH)构建磷饥饿诱导的水稻根系扣除cDNA文库.通过文库筛选和测序获得18个已知基因和47个功能未知基因.这些基因参与了不同的代谢过程,包括磷吸收和转运、信号传导、蛋白质合成和降解、碳水化合物代谢和胁迫反应.Northern杂交结果表明,在磷饥饿胁迫下这些基因呈现不同的表达模式,并且不同代谢过程中的基因对磷饥饿有着不同的反应.     

利用抑制性扣除杂交技术克隆水稻磷饥饿诱导基因

磷素是植物生长所必需的重要元素。在缺磷环境中,植物能够调节自身的形态、生理生化和基因表达水平来适应环境的变化。为研究水稻(Oryzn sativa L.)耐低磷胁迫的分子机理,采用抑制性扣除杂交技术(SSH)构建磷饥饿诱导的水稻根系扣除cDNA文库。通过文库筛选和测序获得18个已知基因和47个功能未知基因。这些基因参与了不同的代谢过程,包括磷吸收和转运、信号传导、蛋白质合成和降解、碳水化合物代谢和胁迫反应。Northern杂交结果表明,在磷饥饿胁迫下这些基因呈现不同的表达模式,并且不同代谢过程中的基因对磷饥饿有着不同的反应。     

国内信息

ＤＥＧＣＭ技术中的ＲＤＡ、ＳＳＨ和ＲＳＤＤ差异表达基因分离技术 (ＤＥＧＣＭ )发展很快 ,主要是ＰＣＲ技术的发展和相应新技术的出现为ＤＥＧＣＭ注入了新的活力。ＰＣＲ引入差式筛选之中 ,可提高效力和降低假阳性率。若引入扣除杂交 ,可发展扣除扩增方案 ,并用特异引物结合于ＰＣＲ中选择扩增差异ｃＤＮＡ而形成代表性差式分析法 (ＲＤＡ)。采用抑制性ＰＣＲ选择性扩增差异基因片段 ,用ＤＮＡ链内“退火”优先于链间退火 ,使非目的序列产生“发夹”式互补结构 ,不能与引物配对 ,从而选择性抑制非目的基因片段的扩增 ,以此形成抑制性差…     

抑制性消减杂交技术在禽类差异表达相关基因筛选中的应用进展

抑制性消减杂交技术(suppression subtractive hybridization,SSH)是目前被广泛用于寻找差异表达基因方面的一种技术,因其具有假阳性率低、灵敏度高、重复性好、特异性强等特点而被大多数研究者所采用。该技术的优势在于可以在转录水平对不同环境、不同生理条件下的组织或细胞进行基因差异表达方面的研究。随着近年来分子生物学的不断发展,对差异表达基因的筛选及克隆已逐渐成为研究的热点。本文主要对抑制性消减杂交技术在鹅、鸭和鸡这三种常见禽类的生产性能、抗病机理以及品种差异等方面研究中的应用进行综述,从而为采用抑制性消减杂交技术研究生命活动的分子作用机制提供更多的参考。     

抑制性差减杂交技术在蚊虫研究中的应用

抑制性差减杂交技术(suppression subtractive hybridization)是在差减杂交和抑制性PCR技术的基础上发展起来的鉴别差异表达基因的实验方法,用于分离2种具有相同或者相似遗传背景,但是表型上有差异的生物样品中差异表达的基因。这项技术在蚊虫许多研究领域,如抗药性、病原体感染、生理特性等得到广泛的应用。本文对抑制性差减杂交技术在蚊虫各研究领域的应用情况进行了综述。     

抑制性消减杂交技术的应用

基因的差异表达是调控各种生命活动的核心分子机制,而分离、克隆并进一步研究差异表达基因已成为现代分子生物学研究的热点,也是功能基因组学研究的重要内容.在研究差异表达基因的诸多项技术中,抑制性消减杂交(SSH)技术具有特异性强、假阳性率低、灵敏度高和快速简便等优点,被广泛地应用于生命科学和医学领域的基因差异表达的研究中.近年来,抑制性消减杂交(SSH)技术得到了相应的改进和完善,而在许多研究领域,该技术在广度和深度上都有了一些新进展.主要就抑制性消减杂交技术的产生背景、原理、技术流程、特点及其最新应用研究进展等方面作简要综述.     

差异表达基因分离技术的研究进展

分离并克隆差异表达基因是生命科学的研究热点.近年来,以差示筛选、扣除杂交等基本方法为基础,先后出现了抑制差减杂交,微阵列技术等多种分析差异表达基因的技术, 使差异表达基因分离方法不断完善.对这些方法的优缺点、发展趋势及应用前景进行了简要综述.     

与芒果子叶切段不定根形成相关基因的cDNA片段的克隆

在研究芒果子叶横切所形成的远轴面和近轴面的不定根形成时发现,只在近轴面形成不定根.该研究利用抑制性扣除杂交(suppres sive subtractivehybridization,SSH)方法,以子叶切段远轴切面作为参照样品,近轴切面作为检测样品,构建正向差异cDNA文库,分离与芒果子叶切段不定根形成相关基因的cDNA片段.经VirtualNorthern杂交分析,获得6个阳性克隆.序列分析和同源性比较结果表明这些cDNA片段均为首次报告,它们分别与转运蛋白、转录调控因子及酶基因的DNA序列同源.     

抑制性消减杂交技术（SSH）及其在基因克隆上的应用

简要概述了抑制性消减杂交技术的基本原理、基本过程、优越性、主要缺陷及其在基因克隆方面的应用进展。     

Epithelial cell kinetics in the descending colon of the rat

Epithelial cell loss was induced in the descending colon of the rat by temporary ischaemia to investigate whether this would lead to an increase in crypt cell proliferation. Shortly after the temporary ischaemia the number of cells per crypt was markedly reduced, and it was shown that the cell loss occurred mainly from the non-proliferating upper half of the crypt. The number of cells per crypt reached control values again after 24-48 h. There was a marked increase in proliferative activity, as reflected by the labelling index after 3HTdR and by the mitotic index, with peak values at 16 and 24 h after ischaemia. After 48 h the proliferative indices were normal again. The increase in crypt cell proliferation was characterized by an increase in the labelling index as well as in the mitotic index per crypt cell position. No enlargement of the proliferative cell compartment in the crypt was observed. It is most likely then that the increase in crypt cell proliferation was brought about by a shortening of the cell cycle, since the growth fraction in the lower half of the crypt approaches 1.0. The possible implications of the present data for the control of colonic cell proliferation and colonic carcinogenesis are discussed.     

小鼠正常肺细胞和肺癌细胞之间的力学特性比较研究

该实验通过鬼笔环肽染色技术观察并比较了小鼠正常肺细胞和肺癌细胞的微丝差异,利用荧光抗体染色技术测定了小鼠单个正常肺细胞和肺癌细胞的α-SMA蛋白含量变化,以及利用CTFM法测定了小鼠正常肺细胞和肺癌细胞的牵引力变化。结果发现,小鼠肺细胞癌变后,细胞内微丝骨架发生了变化,影响了细胞的形态;α-SMA蛋白含量明显下降并且变得分散：细胞投影面积显著减少,大约减少27％,细胞牵引力也显著减小,均方根值大约减少49％。这说明细胞骨架、细胞的形态、α-SMA蛋白和细胞牵引力均与细胞的癌变过程密切相关。     

A dense cell in the epithelium of the gill lamellae of the brook trout, Salvelinus fontinalis (Mitchill)

A cell was found in freshwater brook trout which was similar to a chloride cell as it was mitochondria-rich with an extensive tubular network, but differed in having a dark cytoplasm, large round vesicles in the apical cytoplasm, and large membrane-bound bodies near the nucleus. The base of the cell was separated from the basement membrane by cytoplasm from another epithelial cell. The cell was more rarely found in trout adapted to brackish water and salt water.     

Some factors affecting the chloroplast replication in the mossPlagiomnium trichomanes

Summary Some factors affecting the chloroplast replication were studied using the leaf cells of the mossPlagiomnium trichomanes. There was a significant positive correlation between chloroplast number per cell and cell volume in leaves of any developmental stage. However, when the detached leaves were cultured on nutrient agar, it was observed that the chloroplast replication occurred without cell enlargement regardless of the developmental stage of leaves. This implies that cell enlargement is not an essential factor for the chloroplast replication, but one of the environmental factors affecting it. Light is essential for the chloroplast replication which response to the light intensity. In the dark, there was little increase in chloroplast number per cell. With a light intensity of 50 lux, the increase rate of chloroplast number per cell was about half of that with 3,000 lux. Day length also affected significantly the chloroplast replication.     

Ultrastructural morphology of the nerve cells in the cerebral ganglion of the Acanthocephalan Moniliformis moniliformis

The morphology of the cerebral ganglion of the Acanthocephalan Moniliformis moniliformis was studied in serial sections using electron microscopy. The organization of the cerebral ganglion was typical of other invertebrates with the cell bodies forming a rind, 1 cell thick, and their processes forming the central core of the neuropile. The ganglion was surrounded by a connective tissue capsule composed of collagen-like fibrils. Externally, the free surface of the cell bodies was covered by an electron-dense extracellular lamina. Seventy-six cells were identified in every ganglion examined and, on the basis of their cellular characteristics, they were divided into 5 distinct cell types, classified as type A, B, C, D and E cells. The characteristic morphological features of each cell type have been described, and the distribution of the different cell types in the cerebral ganglion was mapped.     

Towards understanding the control of the division cycle in animal cells.

The author reviewed the historical process by which classical knowledge of cell division accumulated, to give rise to the molecular biology of the cell cycle, and discussed the perspective of this field of research. The study of the control of cell division began at the turn of the century. It was hypothesized that cell division was a physiological regulation necessary for growing cells to maintain a proper nucleocytoplasmic ratio to survive, which was later substantiated by the finding that amoeba cells could be prevented from dividing by repeated excision of the cytoplasm. However, the observation in Tetrahymena that heat-shocked cells grow exceedingly, but fail to divide, suggested that the cell required the accumulation of a labile "division protein" to initiate division. Mechanisms that control the cell cycle were studied in oocytes by nuclear transplantation and cytoplasmic transfer, and in cultured mammalian cells, protozoa, and Physarum plasmodia by cell fusion. These experiments demonstrated the existence of cytoplasmic factors that control the cell cycle. Maturation promoting factor (MPF) thus discovered in frog oocytes became known to be an ubiquitous cytoplasmic factor that causes the transition from interphase to metaphase in all organisms. The insight into the molecular control of cell growth and division was gained from yeast cell genetics. For biochemical analysis of the cell cycle control, the method to observe the cell cycle in vitro was developed using frog egg extracts. Thus, MPF was identified as a cdc2--cyclin protein complex. Its activity was found to depend on synthesis and phosphorylation of these proteins. However, recently it was found that there were cell cycle phenomena that were difficult to explain in these terms. Various other cellular factors, including nucleocytoplasmic ratio and microtubule assembly, were also found to control MPF, as well as the cell cycle. It remained open to future how these factors control MPF to alter the pattern of the cell cycle.     

Extensibility of isolated cell walls in the giant tip-growing cells of the xanthophycean alga Vaucheria terrestris

Apical cell wall fragments isolated from the giant-cellular xanthophycean alga Vaucheria terrestris sensu Götz were inflated with silicone oil by applying internal pressure ranging from 0.1 to 0.7 MPa, and the time-course of cell wall deformation was recorded and analyzed by videomicroscopy. Cell wall extensibility in the tip-growing region was estimated by the pressure required for cell wall extension, the amount of total extension until cell wall rupture and the rate of cell wall extension. Apical cell walls exhibited gradual extension, or creep, during inflation, which was eventually followed by rupture at the apical portion, whereas no appreciable extension was found in the cylindrical basal portion of the cell wall fragment. Besides the largest extension observed around the tip, substantial extension was also observed along the subapical region of the cell wall. The wall extensibility was dependent on the buffer pH used for infiltration before inflation. The optimum pH for the extension was about 8.0, but the cell wall was much less extensible after infiltration with an acidic buffer. Cell wall extensibility was dependent on the pH of the buffer used before inflation, regardless of that used in the previous infiltration. Moreover, pretreatment of the cell wall with a protease caused considerable loosening of cell walls, but affected the pH dependence of cell wall extensibility little. These results indicate that the extensibility of the cell walls in the giant tip-growing cells of the alga is distinct from that of plant cells that exhibit "acid growth" in its dependence on environmental pH and the role of cell wall proteins.     

The kinetics of ox kidney biliverdin reductase in the pre-steady state. Evidence that the dissociation of bilirubin is the rate-determining step.

Four mouse and two human tumour cell lines resistant to alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC), were analysed for the activities of polyamine-biosynthetic and -biodegradative enzymes as well as for cellular polyamine contents. In all but one of these cell lines the resistance to DFMO was based on an overproduction of ODC. In a human myeloma cell line the resistance was based on a greatly enhanced arginase activity. Except for one L1210 variant cell line, all the resistant cell lines contained elevated S-adenosylmethionine decarboxylase activity. Similarly, all the resistant mouse, but not human, cell lines displayed enhanced spermidine and spermine synthase activities. Arginase activity was detected only in human cell lines. In both DFMO-resistant cell lines the activity of arginase was strikingly elevated. Of the biodegradative enzymes, polyamine oxidase activity was readily detectable in all mouse cells, but no measurable activity was found in the human cells. Spermidine/spermine N1-acetyltransferase activity was elevated in three out of four resistant mouse cell lines. Even though the concentration of spermidine was usually lower in the overproducer cells, this was compensated by an increased content of spermine. The two resistant human myeloma cells contained intracellular ornithine concentrations that were from more than 5 to more than 20 times higher than those in the parental cells.     

Epithelial cell kinetics in the descending colon of the rat

The influence of experimental bypass on the epithelial cell kinetics in the rat descending colon was studied. It was found that the number of cells per crypt was markedly reduced at 6 weeks after bypass. The percentage of labelled crypt cells, 1 h after 3HTdR, and the distribution of labelled cells in the crypt was normal. Also the life span of the epithelial cells was the same in control and bypassed colon. The response of crypt cell proliferation to ischaemia-induced cell loss in the bypassed descending colon was similar to the one previously described for normal descending colon. This indicates that the absence of the normal luminal contents does not result in a different response of colonic crypts to induced cell loss. Furthermore, it was found that the number of cells per crypt and the proliferative activity did not change in the transverse colon after temporary ischaemia of the bypassed descending colon. This indicates that the increase in crypt cell proliferation after ischaemia-induced cell loss is a local response.     

Comparison of seven different heterologous protein expression systems for the production of the serotonin transporter

The rat serotonin transporter (rSERT) is an N-glycosylated integral membrane protein with 12 transmembrane regions; the N-glycans improve the ability of the SERT polypeptide chain to fold into a functional transporter, but they are not required for the transmembrane transport of serotonin per se. In order to define the best system for the expression, purification and structural analysis of serotonin transporter (SERT), we expressed SERT in Escherichia coli, Pichia pastoris, the baculovirus expression system and in four different stable mammalian cell lines. Two stable cell lines that constitutively expressed SERT (Imi270 and Coca270) were constructed using episomal plasmids in HEK293 cells expressing the EBNA-1 antigen. SERT expression in the three different inducible stable mammalian cell lines was induced either by a decrease in temperature (cell line pCytTS-SERT), the addition of tetracycline to the growth medium (cell line T-REx-SERT) or by adding DMSO which caused the cells to differentiate (cell line MEL-SERT). All the mammalian cell lines expressed functional SERT, but SERT expressed in E. coli or P. pastoris was nonfunctional as assessed by 5-hydroxytryptamine uptake and inhibitor binding assays. Expression of functional SERT in the mammalian cell lines was assessed by an inhibitor binding assay; the cell lines pCytTS-SERT, Imi270 and Coca270 contained levels of functional SERT similar to that of the standard baculovirus expression system (250,000 copies per cell). The expression of SERT in induced T-REx-SERT cells was 400,000 copies per cell, but in MEL-SERT it was only 80,000 copies per cell. All the mammalian stable cell lines expressed SERT at the plasma membrane as assessed by [3H]-5-hydroxytryptamine uptake into whole cells, but the V(max) for the T-Rex-SERT cell line was 10-fold higher than any of the other cell lines. It was noticeable that the cell lines that constitutively expressed SERT grew extremely poorly, compared to the inducible cell lines whose growth rates were similar to the parental cell lines when not induced. In addition, the cell lines MEL-SERT, Imi270 and T-REx-SERT all expressed fully N-glycosylated SERT and no unglycosylated inactive protein, in contrast to the baculovirus expression system where the vast majority of expressed SERT was unglycosylated and nonfunctional.