汉坦病毒感染细胞整合素受体信号转导相关差异表达基因筛选

通过基因芯片技术观察汉坦病毒感染细胞后整合素受体信号转导相关基因的差异表达,以探讨汉坦病毒进入细胞的分子机制.用汉滩病毒76-118株感染原代人胚肺成纤维细胞,同时设立正常对照细胞,于感染后第6h、24h和96h同时抽提感染组与正常对照组细胞总RNA,利用荧光标记dUTP逆转录制备cDNA探针,与人类受体及信号转导类表达谱芯片杂交,并对Cy3、Cy5荧光信号做扫描分析.结果显示有37项基因出现差异表达,其中31项基因在感染后6h出现差异表达,13项下调、18项上调;但在感染后24h和96h分别只有5项和4项基因出现表达差异;而且整合素信号转导通路中的重要基因如一些蛋白激酶相关基因在感染6h表达增强,丝化增强子1在感染6h和96h表达下调,微管相关蛋白1A在感染6h表达上调、感染24h和96h表达下调.这些进一步说明汉坦病毒感染细胞与整合素有关.     

H2O2诱发人成纤维细胞衰老样变化的基因表达谱

以 5 0 μmol/LH2 O2 作用体外培养的人胚肺二倍体成纤维细胞 4次 ,使之出现不可逆的衰老表型 .提取年轻细胞及H2 O2 处理早老细胞的mRNA ,以荧光物Cy3标记年轻细胞cDNA ,Cy5标记H2 O2 处理的细胞cDNA ,并与点有 40 96条人类基因的芯片杂交 ,利用计算机数据处理判断基因是否存在表达差异 .结果显示 :有 12 3种基因的表达变化较显著 ,这些基因参与细胞周期进程、细胞代谢及蛋白质修饰、细胞外基质及细胞骨架蛋白的形成和调节、炎症反应、调节受体酪氨酸蛋白激酶和G蛋白耦联受体信号转导 .     

鸭肠炎病毒感染对鸭肠道黏膜组织转录组变化分析

为了探究鸭肠炎病毒感染对鸭十二指肠黏膜组织相关免疫基因的变化影响,本试验采用鸭肠炎病毒接种35日龄的健康樱桃谷鸭,将其分为3组(2个试验组和1个对照组),在感染后24 h、48 h采集十二指肠,提取总RNA进行浓度、纯度及完整性检测后构建cDNA文库,使用BGISEQ-500平台对2个实验组和对照组黏膜组织测序,筛选差异表达基因,并用GO和KEGG数据库进行分析。结果显示,2个实验组感染鸭肠炎病毒后T24和T48分别筛选出共720个差异表达基因,其中T24表达上调58个,下调163个;T48表达上调77个,表达下调422个。GO功能注释结果显示,感染后差异基因都与代谢、免疫、炎症和调控途径等密切相关。KEGG通路富集分析发现,感染T24的差异表达基因富集到神经活性配体-受体相互作用、补体和凝血级联、氮代谢、cAMP信号通路和IL-17信号转导通路;感染T48的主要差异表达基因富集到神经活性配体-受体相互作用、细胞因子-细胞因子-受体相互作用、趋化因子信号通路、造血细胞谱系和补体和凝血级联。借助BGISEQ-500平台对鸭肠炎病毒感染后十二指肠黏膜组织相关因子的变化来探究其致病机制及其他生物学通路,弥补了非模式生物转录组研究中缺乏基因信息的不足。     

汉坦病毒受体

汉坦病毒感染可引起两种严重威胁人类生命的传染性疾病:肾综合征出血热(HFRS)和肺综合征出血热(HPS)[1,2],这两种疾病都与急性血小板减少和血管渗透性变化有关[3].因此,弄清汉坦病毒与宿主细胞的相互作用及其机制,对控制汉坦病毒性疾病显得尤为重要.整合素对维持血管壁的渗透性和完整性具有重要作用.一种整合素可以在不同的细胞上表达,同一种细胞也可以表达多种不同的整合素,并且不同类型整合素的作用也各有不同.在汉坦病毒与内皮细胞相互作用研究中,细胞整合素受体起到了关键性的作用,致病性汉坦病毒通过细胞膜上β3整合素感染细胞,而非致病性汉坦病毒通过细胞膜上β1整合素感染细胞[4,5].这些都为汉坦病毒的受体和发病机制研究奠定了一定的基础.本文拟对该研究近况作一综述,为汉坦病毒致病机制的深入研究提供参考.     

应用抑制消减杂交和cDNA芯片技术筛选流行性感冒病毒感染相关基因

病毒基因组有限的编码能力和以病毒蛋白为靶的抗病毒药物易出现耐药性,使从病毒感染宿主筛选病毒感染相关生物大分子作为抗病毒药靶和诊断标志物成为新的研究方向。为了筛选流行性感冒（流感）病毒感染相关基因,采用抑制消减杂交（suppression subtractive hybridization,SSH)技术,以流感病毒A／鲁防／93－9（H3N2）感染的MDCK细胞及正常MDCK细胞为材料,构建病毒感染特异性差减cDNA文库。从文库中随机挑取约800个克隆,PCR扩增其中插入片段,经纯化、紫外定量后,用基因芯片自动点样仪点在氨基片上,制备cDNA芯片。将流感病毒感染的MDCK细胞和正常MDCK细胞的总RNA分别用Cy3、Cy5反转录荧光标记后,与cDNA芯片杂交,用芯片扫描仪扫描获得芯片杂交信号,经阳性对照校正和归一化处理后,以如下条件作为判定基因差异表达的标准;（a)Cy3与Cy5的信号比值大于1．5（正常细胞用Cy5标记）或小于0．67（正常细胞用Cy3标记）;（b)Cy3和Cy5信号值之一必须大于1000。经cDNA芯片筛选获得了18个流感病毒感染特异性克隆,经测序和生物信息学分析发现均为流感病毒感染相关新基因EST。流感病毒感染相关基因cDNA片段的获得,为新型病毒药靶诊断标志物发现和功能研究提供了基础。     

二烯丙基二硫作用于K562细胞后凋亡相关基因的差异表达

目的:研究二烯丙基二硫(diallyl disulfide DADS)作用于人白血病K562细胞后凋亡相关基因的差异表达,为进一步探讨DADS诱导K562细胞凋亡的分子机制提供基础.方法:采用瑞士-吉姆萨和Hoechst33342染色观察细胞形态学变化.运用基因芯片技术检测40 mg/LDADS作用于K562细胞24h后凋亡相关基因的差异表达,选择其中上调基因GADD45A、下调基因HO-1运用RT-PCR技术进行验证.结果:40mg/L的DADS作用于K562细胞后出现凋亡所具有的典型形态学变化.40 mg/L DADS作用K562细胞24 h后有14个凋亡相关差异表达基因.GADD45A、HO-1基因表达情况与基因芯片结果一致.结论:DADS可能通过多个基因和多条信号转导通路共同作用诱导人白血病细胞K562凋亡.     

H2O2诱发人成纤维细胞衰老样变化的基因表达谱

以50 μmol/L H₂O₂作用体外培养的人胚肺二倍体成纤维细胞4次,使之出现不可逆的衰老表型.提取年轻细胞及H₂O₂处理早老细胞的mRNA,以荧光物Cy3标记年轻细胞cDNA,Cy5标记H₂O₂处理的细胞cDNA,并与点有4 096条人类基因的芯片杂交,利用计算机数据处理判断基因是否存在表达差异.结果显示：有123种基因的表达变化较显著,这些基因参与细胞周期进程、细胞代谢及蛋白质修饰、细胞外基质及细胞骨架蛋白的形成和调节、炎症反应、调节受体酪氨酸蛋白激酶和G蛋白耦联受体信号转导.     

人体肝癌细胞急性低氧及低氧习服差异表达基因分析

本文分析了人体肝癌细胞(HepG2)急性低氧处理以及低氧习服处理后基因表达谱的改变。急性低氧处理为细胞在1％氧气中培养48h,低氧习服处理为细胞在1％氧气中培养24h,常氧培养24h,以此作为一个周期,重复6个周期。联合应用抑制消减杂交技术和cDNA芯片技术,筛选HepG2细胞经急性低氧处理与正常培养细胞相比差异表达的基因,以及经低氧习服处理细胞与正常培养细胞相比差异表达的基因。结果显示,HepG2细胞经急性低氧处理与在常氧条件下培养相比,差异表达的基因有37个,表达水平全部表现为下调,其中包括参与细胞周期、细胞应激、细胞信号转导、细胞骨架形成、转录相关蛋白及细胞代谢相关蛋白的基因,1个未知基因序列、4个EST序列、5个线粒体蛋白基因,另外有功能不明的蛋白质基因12个。低氧习服处理的细胞与常氧条件下培养的细胞相比,差异表达的基因有6个,其中包括两个线粒体蛋白基因、金属蛋白酶1基因、转铁蛋白基因、Thymosin ．beta-4和TPT1基因。其中线粒体蛋白ND4、转铁蛋白、Thymosin.beta-4和TPT1基因的表达呈上调,线粒体NDl及金属蛋白酶1基因的表达水平呈下调。经低氧习服处理后,细胞低氧耐受力提高,低氧习服处理细胞基因的表达与急性低氧处理细胞和正常培养细胞的基因表达不同,这种变化可能与低氧习服细胞低氧耐受力的增强有关。     

应用基因表达谱芯片从不同转移能力的 乳腺癌细胞中筛选转移相关基因

人乳腺癌细胞系 MCF-7 及其转移亚克隆 LM-MCF-7 为肿瘤转移分子机制的研究提供了细胞模型 . 应用基因芯片技术比较两种具有不同转移能力细胞系基因表达谱的差异,寻找乳腺癌转移相关基因 . 提取两种细胞总 RNA ,分别用 Cy5-dCTP 、 Cy3-dCTP 标记 LM-MCF-7 和 MCF-7 的 cDNA ,并与含有 21 329 个基因的芯片进行杂交并扫描,利用 GenePix Pro 4.0 图像分析软件处理数据判断基因是否在两个细胞中存在表达差异 . 经互换荧光标记物重复两次实验,共筛选出差异表达基因 67 个,其中 41 个在 LM-MCF-7 细胞中表达上调, 26 个在 LM-MCF-7 细胞中表达下调 . 应用实时定量 RT-PCR 对 7 个表达差异明显的基因进行了验证 . 生物信息学分析结果提示,上述发现的差异基因编码产物与细胞内信号转导、转录调节、应激反应、新陈代谢、发育、细胞运动、细胞凋亡和细胞粘连等功能有关 . 据文献报道,这些差异表达的基因中有 35 个与肿瘤有关,其中 9 个与乳腺癌转移有关, 6 个可能参与肿瘤浸润和转移过程 . 根据基因芯片检测的结果,从功能上对 LM-MCF-7 细胞和 MCF-7 细胞与细胞凋亡的关系进行了研究,发现具有高转移倾向的 LM-MCF-7 细胞与 MCF-7 细胞相比,抗凋亡能力较强 . 上述与肿瘤转移相关基因在肿瘤转移中的作用及其分子机理有待深入研究 .     

水菠菜叶乙醇提取物对MG-63骨质疏松相关基因表达的影响

为探讨水菠菜叶乙醇提取物与抗骨质疏松作用的关系及其作用机制,用水菠菜叶乙醇提取物处理人成骨样细胞MG-63 48 h后,提取水菠菜叶乙醇提取处理组和对照组细胞的总RNA,将两组RNA纯化为mRNA,逆转录成cDNA,用Cy3和Cy5两种不同的荧光染料进行线性扩增标记,然后与骨质疏松相关基因表达谱芯片杂交,扫描后对获得的数据用LuxScan 3.0软件分析.研究发现经水菠菜叶乙醇提取物诱导后MG-63细胞的244个骨质疏松相关基因中,有c-Fos、JNK、ODF、c-src、OSCAR、RANKL、Samd4、Samd6、Samd7 9个基因表达明显上调,LRP5、Dkk1、TGFB、OPG 4个基因明显下调.结果表明水菠菜叶乙醇提取物能改变骨质疏松相关基因的表达,抗骨质疏松作用机制可能与Wnt/β-catenin蛋白信号传导途径和骨保护素/核因子受体激活信号传导途径有关,与雌激素内分泌途径无关.     

六种藓类植物茎的比较解剖学研究

通过对6种藓类植物,即褶叶青藓(Brachythecium salebrosum(Web.et Mohr.)B.S.G.)、湿地匐灯藓(Plagiomnium acutum(Lindb.)Kop.)、侧枝匐灯藓(Plagiomnium maximoviczii(Lindb.)Kop.)、大凤尾藓(Fissidensnobilis Griff.)、大羽藓(Thuidium cymbifolium(Doz.et Molk.)B.S.G.)和大灰藓(Hypnum plumaeforme Wils.)嫩茎和老茎的石蜡切片和显微观察发现,同一藓类植株的嫩茎和老茎,茎结构稳定,不同种藓类植物茎横切面具有不同特征.植物体茎横切面形状、表层细胞的层数、细胞大小和细胞壁厚薄、皮层细胞大小和形状、中轴的有无以及比例等特征可以作为藓类植物的分科分类依据之一.     

Cause of Senescence of Nine Sorts of Flowers

The levels of endogenous phytohormones and respiratory rate in nine sorts of flowers such as Cymbidium faberi Rolfe, Nopalxochia ackermannii Kunth and others were investigated both at full bloom and senescence and meanwhile the effect of exogenous phytohormones on prolonging the blossoms and promoting ethylene production were tested. There is a high content of endogenous ethylene in all the long-lived flowere, about 3–16 folds higer than the short-lived ones. There is a high level of ABA at full blooming flowers of short-lived flowers, in which there is no or only some cytokinins in it, but the ratio of CTK (6BA+zeatin)/ABA is smaller(l.7). The endogenous ABA reached a much higher level at senescence in all nine sorts of flowers, so it is reasonable to consider that it is ABA which plays an important role of regulation in controlling flower's senescence. There is a much higher level of GA3 and zeatin in the long-lived flowers which is not demonstrated in the shortlived ones. The respiratory rate is one of the factors controtling the longevity of flowers, but it does not play a decided role. Application of 6BA and zeatin prolongs distinctly orchid’s longevity, however exogenous IAA through the promotive action on ethylene production, evidently extends the longevity of the flowers of the Nopalxochia ackermannii Kunth.     

Photoregulation of seed germination of wild-type and of an aurea-mutant of tomato

Seed germination of an aurea mutant of tomato ( Lycopersicon esculentum Mill.) is promoted by continuous irradiation with red, far-red or long-wavelength far-red (758 nm) light as well as by cyclic irradiations (5 min red or 5 min far-red/25 min darkness). Far-red light applied immediately after each red does not change the germination behaviour. Seed germination of the isogenic wild-type, cv. UC-105, is promoted by continuous and cyclic red light while it is inhibited by continuous and cyclic far-red light and by continious 758 nm irradiation. Far-red irradiation reverses almost completely the promoting effect of red light. The promoting effect (in the aurea mutant) and the inhibitory effect (in the wild-type) of continuous far-red light do not show photon fluence rate dependency above 20 nmol m⁻² s⁻¹. It is concluded that phytochrome controls tomato seed germination throgh low energy responses in both the wild type and the au mutant. The promoting effect of continuous and cyclic far-red light in the au mutant can be attributed to a greater sensitivity to P_fr.     

真菌类遗传学分析的知识结构教学

本文以认知结构理论为指导,讨论了真菌类遗传分析与高等动植物遗传分析的内在联系,认为利用这种内在联系进行教学可收到好的效果并说明了作者的具体教学过程。 Abstract:In the paper, the relationship between genetic analysis of Fungi and genetic analysis of high animal and plant was discussed.A good results were obtained when we adopted this method in the teaching.     

龙胆科药用植物化学成分的研究现状

龙胆科植物在我国的分布范围很广,且多数为药用植物,其多数种属的药用植物,至今其化学成分尚未被系统研究。综述了目前龙胆科药用植物的化学成分的研究现状及一般提取方法,对近年来发现的环烯醚萜及裂环烯醚萜类化合物进行了总结,为本科药用植物的更深入研究提供了参考。     

The effects of glutamine of the maintenance of embryogenic cultures of Cryptomeria japonica

Summary Embryogenic tissues of sugi (Cryptomeria japonica) were induced on a modified Campbell and Durzan (CD) medium containing 1 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 600 mg l⁻¹ glutamine, and subcultured in the medium of the same composition for over 1 yr. This resulted in a mixed culture of embryogenic and non-embryogenic cells. When embryogenic cells were isolated and cultured independently, their capacity to form embryogenic aggregates was lost. Thus, the non-embryogenic cells present within a mixed culture system were essential to the formation of embryogenic aggregates. When embryogenic tissues were isolated and cultured independently on a high glutamine-containing (2400 mg l⁻¹) medium, dry weights and endogenous levels of glutamine increased, and the tissue could generate a large number of embryogenic aggregates. Amino acid analysis of embryogenic and non-embryogenic cells from the maintenance culture indicated a higher level of glutamine was present in the latter. The high endogenous level of glutamine in the non-embryogenic portion of mixed cell masses may be the supplier of glutamine for maintaining the embryogenic property of the tissues.     

Specificity of action of piperidylcholine derivatives as substrates of cholinesterases of various origin

The review deals with study of enzymologic properties of a novel highly specific acetylcholinesterase substrate, N-(β-acetoxyethyl) piperidinium iodomethylate (“piperidylcholine”), and its 30 derivatives that were tested as effectors of cholinesterases of mammals and various species of Pacific squids. It was proven for the first time that responsible for specificity of action was structure of cyclic ammonium grouping of the alcohol part of molecule of the ester substrate. Analysis of specificity is performed based on enzymatic hydrolysis parameters—activity of catalytic center of cholinesterases and bimolecular constant of the reaction rate that are determined at optimal and low substrate concentrations. Among the specially synthesized group of thioester compounds there is revealed one more highly specific acetylcholinesterase substrate—N-(β-acetoxyethyl) piperidinium.     

Interconnection of parameters of regulation system of lipid peroxidation and of morphophysiological parameters of mouse liver

A complex analysis of seasonal fluctuations of the mean group parameters of the system of regulation of lipid peroxidation has been performed in liver of Balb/c mice. Association of lipid characteristics and morphophysiological parameters is studied in the Balb/c mouse liver. An inter-connection is revealed between the liver index and the amount of lysoforms of phospholipids, the scale and character of the interconnection differing essentially depending on proportion of phos-phatidylcholine in mouse liver phospholipids.