siRNA沉默SIRT1基因对宫颈癌细胞HeLa增殖和凋亡的影响

化学合成靶向SIRT1基因的小干扰RNA,脂质体法转染人宫颈癌细胞株HeLa,观察小干扰RNA沉默SIRT1基因对HeLa增殖及细胞凋亡的影响。在优化siRNA SIRT1转染条件的基础上,应用RT-PCR和Western blot分别检测各组SIRT1 mRNA、SIRT1蛋白及凋亡相关蛋白的表达;CCK-8法检测细胞增殖抑制率;Hoechst荧光染色法和流式细胞仪检测细胞凋亡。结果表明,siRNA SIRT1转染细胞组SIRT1 mRNA水平和蛋白表达量明显低于对照组;siRNA SIRT1转染组细胞增殖受抑制,细胞凋亡率明显增加;凋亡相关蛋白P53、P21表达上调,Survivin表达下调。上述结果表明:siRNA SIRT1诱导的HeLa细胞凋亡与P53、P21、Survivin通路关系密切,但siRNA SIRT1诱导HeLa细胞凋亡的详尽机制有待进一步研究。     

KDR靶向RNA干扰对MCF一7细胞凋亡的影响

目的：研究KDR靶向RNA干扰对MCF-7细胞凋亡的影响,探讨其可能的机制。方法：采用阳离子脂质体Lipofecta．mine2000TM作为转染试剂将人KDR基因的siRNA转染人类乳腺细胞株MCF-7,诱RNAi,采用Hoechst33258染色和半定量RT—PCR检测Caspase-3、survivin的mRNA表达及细胞凋亡变化;比色法检测Caspase．3的活性;利用免疫组织化学方法检测survivin的表达,并用图像分析仪分析蛋白表达强度。结果：靶向KDR的siRNA转染MCF-7后,Caspase-3的mRNA表达上调,survivin基因mRNA及蛋白表达水平下调（P〈0．05）。结论：KDRsiRNA通过减少乳腺癌细胞survivin的表达,增加Caspase．3表达来促进肿瘤细胞凋亡,发挥其抗肿瘤作用。     

siRNA沉默Survivin基因对鼻咽癌CNE-2细胞凋亡的影响

合成Survivin小干扰RNA(small interfering RNA,siRNA)序列,用于转染人鼻咽癌细胞株CNE2,体外观察Survivin siRNA转染后CNE-2细胞Survivin mRNA、蛋白的表达、细胞增殖和凋亡情况。设计合成3段特异性Survivin siRNA,用siRNA转染鼻咽癌CNE-2细胞株,设置空白对照和阴性对照组,通过Real-time PCR检测CNE-2细胞的Survivin mRNA相对表达量,Western blotting检测Survivin蛋白的表达,流式细胞术及原位细胞凋亡检测转染后细胞凋亡情况。siRNA转染CNE-2细胞后,siRNA 1组和3组mRNA表达抑制率分别为(68.46±7.94)%、(49.44±3.78)%,siRNA 2组结果无显著差异(p0.05)。蛋白相对表达量只有siRNA 1组降低,表达抑制率为(30.61±2.47)%,流式细胞术和原位细胞凋亡检测转染后CNE-2细胞数量明显降低,细胞凋亡比例增高。siRNA干扰沉默Survivin基因能有效抑制CNE-2细胞的增殖和诱导细胞凋亡。本研究为Survivin基因可能作为治疗鼻咽癌的一个靶点,为Survivin基因沉默可能是治疗鼻咽癌高效的途径提供体外实验支持。     

小干扰RNA靶向VEGF基因体内外抑制乳腺癌细胞MCF-7的增殖

 血管生成与肿瘤生长、侵袭、转移密切相关.血管内皮生长因子能特异地促进内皮细胞分裂、增殖及迁移,在肿瘤新生血管生成过程中起着至关重要的作用.通过RNAi抑制VEGF表达的抗血管生成疗法可有效应用于肿瘤治疗.本研究采用化学修饰的siRNA在体内外抑制VEGF基因表达,探讨化学修饰的siRNA介导的RNA干扰技术在乳腺癌基因治疗的可行性和特异性.选用阳离子脂质体Lipofectamine^TM2000作为转染试剂,将针对人VEGF基因的小干扰RNA(small interfering RNA,siRNA)转染人类乳腺细胞株MCF-7和裸鼠移植瘤,在体内外诱导RNAi.采用四甲基偶氮唑蓝（MTT）法,逆转录聚合酶链反应(RT-PCR),蛋白印迹实验等检测siRNA治疗组和对照组VEGF基因表达及细胞增殖变化.体外实验结果显示：靶向VEGF基因siRNA转染乳腺癌MCF-7细胞后,细胞生长抑制率达52.5%;VEGF的mRNA和蛋白表达水平显著降低（P<0.01）;裸鼠体内实验结果显示：siRNA治疗组瘤组织的增长受到明显抑制;RT-PCR结果同时表明治疗组VEGF表达下调.体内外对照组各指标无显著变化.化学修饰的siRNA介导的RNAi在体内外均能成功下调靶基因VEGF的表达,抑制MCF-7细胞增殖,是潜在的肿瘤治疗新方法.     

应用siRNA技术探讨MCF-7乳腺癌细胞分泌的VEGF对树突状细胞的影响

为探讨MCF-7乳腺癌细胞分泌的血管内皮生长因子（ vascular endothelial growth factor, VEGF）对树突状细胞（dendritic cell, DC）功能及其分化的影响,针对VEGF基因设计siRNA（small interfering RNA, siRNA),采用脂质体转染法以100 nmol/L最佳转染浓度导入MCF-7乳腺癌细胞（siRNA组）,以脂质体Lipofectamine　2000TM转染MCF-7 乳腺癌细胞培养上清培养正常DC作为对照（对照组）,采用ELISA法检测经siRNA 干扰VEGF基因后的MCF-7 乳腺癌细胞分泌的VEGF因子含量, Western 印迹检测VEGF蛋白表达,以探讨siRNA的基因沉默效果;以siRNA组和对照组培养上清分别培养外周血单个核细胞,用流式细胞仪检测所诱导DC表型CD1a、CD80、CD83、CD86和HLA-DR的表达,用MTT法检测转染前后两组DC 诱导的细胞毒性T淋巴细胞（cytotoxic T lymphocyte, CTL）对MCF-7细胞的细胞毒作用.结果显示,MCF-7 乳腺癌细胞培养上清能明显抑制正常DC分化成熟及抗原递呈能力,干扰VEGF基因后MCF-7 乳腺癌细胞培养上清对DC的影响明显降低,CD80、CD83、CD86和HLA-DR的表达较对照组显著升高,而CD1a表达下降（P＜0.01）.转染前后DC 诱导的CTL对MCF-7细胞的杀伤活性有明显差异（P＜0.01）.由此可见,siRNA可靶向抑制MCF-7乳腺癌细胞VEGF的表达,下调VEGF后的MCF-7 细胞上清对DC分化成熟及功能的抑制作用明显降低,从而推测VEGF在肿瘤的发生、发展和免疫抑制方面可能起着重要的作用.     

下调CENP-E对乳腺癌MCF-7细胞的影响

目的:探讨有丝分裂检查点蛋白着丝粒蛋白-E(CENP-E)基因在肿瘤发生发展中的作用。方法:利用shRNA下调CENP-E基因的表达,分别用巢式PCR和Western blot检测CENP-E mRNA和蛋白的表达;MTT检测CENP-E下调后MCF-7细胞的增殖变化;流式细胞术检测CENP-E下调后对MCF-7细胞凋亡的影响;Transwell试验检测MCF-7细胞的迁移和侵袭能力变化;间接免疫荧光检测细胞内CENP-E蛋白和有丝分裂情况。结果:shRNA能有效抑制CENP-E mRNA和蛋白的表达。MTT结果显示CENP-E下调后MCF-7细胞的增殖能力减弱(P<0.05);流式细胞术显示下调CENP-E后能促进MCF-7细胞的凋亡;间接荧光结果显示CENP-E干扰后MCF-7细胞内CENP-E蛋白减少并伴有核分裂异常;Transwell试验显示CENP-E干扰组细胞的迁移和侵袭能力增强(P<0.05)。结论:下调部分CENP-E的表达能抑制MCF-7细胞的增殖,促进MCF-7细胞的凋亡,增强MCF-7细胞的迁移和侵袭能力。     

RNA干扰NUP88基因对人乳腺癌MCF-7细胞生长及侵袭力的影响

目的： 构建重组慢病毒介导的NUP88-shRNA载体,通过RNAi技术分别观察沉默NUP88后对MCF-7增殖,粘附,侵袭和转移情况的影响,为乳腺癌的临床基因治疗寻找新的靶点。方法： 构建NUP88重组慢病毒表达载体,包装后检测滴度,以最佳复感染指数转染乳腺癌MCF-7细胞,利用RT-PCR和Western blot检测各组MCF-7细胞中mRNA和蛋白的表达效率;MTT法和流式细胞仪检测法,检测NUP88基因被干扰后对MCF-7细胞增殖和凋亡的影响;细胞侵袭实验检测NUP88基因被干扰后对MCF-7侵袭力的影响。结果 四组病毒及一组阴性对照均构建成功,滴度均为4E+8TU/ml;RT-PCR和Western blot检测,结果表明：经NUP88-shRNA转染的MCF-7细胞组NUP88 mRNA和蛋白质的表达与经阴性转染组和空白MCF-7细胞组相比,差异明显具有统计学意义（P<0.01）;测定NUP88-shRNA1组沉默效率最高,沉默率可达到86%;MTT法结果表明：实验组经NUP88-shRNA1慢病毒转染后细胞增殖程度显著减少,与空白组和对照组相比有显著性差异（P<0.05）。流式细胞仪检测三组MCF-7细胞凋亡结果表明：实验组经慢病毒转染后细胞凋亡率显著增加,与对照组和空白组相比有显著性差异（P<0.05）;细胞侵袭实验表明：在肿瘤细胞常规培养24h后,实验组与空白组和阴性对照组比较,穿膜细胞数量明显减少,具有显著性差异（P<0.05） 结论： NUP88重组慢病毒可以通过RNAi成功抑制MCF-7中NUP88基因的表达,并能显著抑制其增殖及远处的侵袭能力。     

沉默FBI-1基因对三阴性乳腺癌细胞MDA-MB-231增殖和凋亡的影响

本研究旨在观察短发夹状RNA (short hairpin RNA, shRNA)介导的人类免疫缺陷病毒短转录诱导物连接因子1 (FBI-1)基因沉默对三阴性乳腺癌细胞MDA-MB-231增殖和凋亡的影响。用qRT-PCR和Western blot分别检测FBI-1、Bcl-2、Bax、cleaved-Caspase3和Survivin的mRNA和/或蛋白的表达水平;采用shRNA干扰技术沉默MDA-MB-231细胞中FBI-1基因的表达;用CCK-8法以及克隆形成实验检测细胞增殖;用流式细胞术检测细胞凋亡;用裸鼠皮下成瘤实验检测细胞的成瘤能力。结果显示,MDA-MB-231细胞的FBI-1 mRNA和蛋白的表达水平明显高于正常乳腺上皮细胞MCF-10A;经shRNA靶向沉默FBI-1基因表达后,细胞增殖能力明显降低,细胞凋亡率显著增高,伴随Bcl-2和Survivin蛋白表达明显下调、Bax蛋白表达显著上调和Caspase 3活化,MDA-MB-231细胞的裸鼠皮下成瘤能力受抑制。以上结果提示,靶向沉默FBI-1基因表达可以抑制MDA-MB-231细胞增殖,诱导细胞凋亡并抑制细胞的裸鼠皮下成瘤能力。     

抑制VEGF基因表达对乳腺癌细胞细胞周期的影响

目的：研究针对VEGF基因的siRNA（small interferenceRNA）对乳腺癌MCF-7细胞细胞周期的影响。方法：依据Promega公司在网上提供的设计软件,设计针对VEGF基因的siRNA,合成DNA模板,体外转录合成siRNA。脂质体转染法将合成的siRNA转染入MCF-7细胞,以未转染细胞以及错义序列siRNAscr转染细胞为对照。用细胞计数法检测siRNA对MCF-7细胞增殖的影响：流式细胞法检测细胞周期变化,RT—PCR法比较转染前后p21、CyclinDl表达水平的变化,Westemblot检测转染前后磷酸化ERK的表达。结果：细胞计数法结果显示,转染24h后siRNA明显抑制MCF-7细胞增殖,转染48h后,抑制效率稳定。siRNA转染后能有效地抑制MCF-7细胞的增殖,阻滞细胞周期于G0／G1期,S期细胞明显减少,G0／G1期细胞比例逐渐增多;p21mRNA表达显著上调,抑制CyclinD1mRNA及磷酸化ERK蛋白的表达。结论：体外转录合成的siRNA可能通过上调细胞周期蚤白激酶抑制剂p21的表达,下调CyclinDl及磷酸化ERK的表达,将细胞周期阻滞于G0／G1期,从而显著抑制MCF-7细胞的增殖。     

干扰PGI调控HIF-1α的表达以及促进乳腺癌细胞凋亡

本实验探究乳腺癌MCF-7细胞中干扰PGI的表达对HIF-1α的调控以及对细胞凋亡的影响。首先,使用慢病毒质粒转染构建干扰PGI(PGI-si RNA)的稳转细胞株,然后使用Hoechst33258染色和流式细胞术(FCM)检测细胞的凋亡,随后,采用RT-PCR检测PGI、HIF-1α和LDHA基因的表达,Western blotting检测Bcl-2、Bax、PGI、LDHA、HIF-1α和ERK蛋白的表达。成功构建PGI-si RNA的MCF-7稳转细胞株后,Hoechst33258染色实验结果显示,PGI-si RNA稳转细胞株中出现核固缩、染色质凝集等形态学改变,FCM结果亦表明PGI-si RNA稳转细胞株发生细胞凋亡。RT-PCR结果显示,PGI-si RNA稳转细胞株中PGI、LDHA和HIF-1α基因表达下调;Western blotting结果显示,PGI-si RNA稳转细胞株中LDHA、HIF-1α、ERK和Bcl-2蛋白表达下调,Bax蛋白表达上调。以上实验结果表明:在乳腺癌MCF-7细胞中,干扰PGI可抑制HIF-1α的表达,同时促进MCF-7细胞凋亡。     

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.     

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.     

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.     

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。     

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.     

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.     

Physiological characteristics of various species of strains of carboxydobacteria

Seven strains of aerobic carbon monoxide-oxidizing bacteria (carboxydebacteria) when growing on CO as sole source of carbon and energy had doubling times which ranged from 12–42 h. The activity profiles obtained after discontinuous sucrose density gradient centrifugation indicated that the CO-oxidizing enzymes are soluble and the hydrogenases are membrane-bound in all strains examined. The CO-oxidizing enzymes of Pseudomonas carboxydohydrogena, Pseudomonas carboxydoflava, Comamonas compransoris, and the so far unidentified strains OM2, OM3, and OM4 had a molecular weight of 230,000; that of Achromobacter carboxydus amounted to 170,000. The molecular weights of the CO-oxidizing and H₂-oxidizing enzymes turned out to be identical. The cell sonicates were shown to catalyze the oxidation of both CO and H₂ with methylene blue, thionine, phenazine methosulfate, toluylene blue, dichlorophenolindophenol, cytochrome c or ferricyanide as electron acceptors. Methyl viologen, benzyl viologen, FAD⁺, FMN⁺, and NAD(P)⁺ were not reduced. The spectrum of electron acceptors was identical for all strains tested. Neither free formate, hydrogen nor oxygen gas were involved in the CO-oxidation reaction. Methylene blue was reduced by CO at a 1:1 molar ratio. The results indicate that CO-oxidation by carboxydobacteria is catalyzed by identical or similar enzymes and that the reaction obeys the equation CO+H₂OCO₂+2H⁺+2e^- as previously shown for Pseudomonas carboxydovorans.Dedicated to Otto Kandler remembering almost three decades of enjoyable cooperation     

Modulation of allostery of pyruvate kinase by shifting of an ensemble of microstates

Since the introduction of the concepts of allostery about four decades ago, much advancement has been made in elucidating the structure-function correlation in allostery. However, there are still a number of issues that remain unresolved. In this review we used mammalian pyruvate kinase (PK) as a model system to understand the role of protein dynamics in modulating cooperativity. PK has a triosephosphate isomerase (TIM)(α/β)₈ barrel structural motif. PK is an ideal system to address basic questions regarding regulatory mechanisms about this common (α/β)₈ structural motif. The simplest model accounting for all of the solution thermodynamic and kinetic data on ligand-enzyme interactions involves two conformational states, inactive E^T and active E^R. These conformational states are represented by domain movements. Further studies provide the first evidence for a differential effect of ligand binding on the dynamics of the structural elements, not major secondary structural changes. These data are consistent with our model that allosteric regulation of PK is the consequence of perturbation of the distribution of an ensemble of states in which the inactive E^T and active E^R represent the two extreme end states. Sequence differences and ligands can modulate the distribution of states leading to alterations of functions. The future work includes: defining the network of functionally connected residues; elucidating the chemical principles governing the sequence differences which affect functions; and probing the nature of mutations on the stability of the secondary structural elements, which in turn modulate allostery.