汉黄芩素抗骨肉瘤143B细胞的实验研究

目的:观察汉黄芩素对人骨肉瘤细胞系143B增殖和凋亡的影响,并探讨其可能的作用机制。方法:采用体外培养人成骨肉瘤细胞系143B,CCK-8实验检测不同浓度汉黄芩素对骨肉瘤143B细胞增殖抑制作用;流式细胞术分析汉黄芩素对癌细胞周期分布及凋亡的影响;Western Blot检测凋亡相关蛋白Bax、Bcl-2、cleaved caspase-9和cleaved caspase-3的表达水平。结果:CCK-8结果显示汉黄芩素以时间、浓度依耐性的方式抑制骨肉瘤143B细胞的增殖;流式细胞术结果表明汉黄芩素可导致骨肉瘤细胞周期阻滞于G0/G1期并以浓度依赖的方式诱导骨肉瘤细胞凋亡;Western Blot检测结果证明,汉黄芩素可上调骨肉瘤细胞中促凋亡蛋白Bax、cleaved caspase-9、cleaved caspase-3的表达,而下调抑制凋亡蛋白Bcl-2。结论:汉黄芩素抑制骨肉瘤细胞增殖、导致细胞周期阻滞,促进其凋亡,并呈现时间和浓度依赖性,汉黄芩素激活Caspase凋亡途径及诱导细胞周期阻滞可能是其抗骨肉瘤的作用机制。     

甲基莲心碱抗乳腺癌 MCF-7细胞的研究

目的:观察甲基莲心碱对乳腺癌细胞系MCF-7增殖和凋亡的影响,并探讨其诱导乳腺癌细胞系MCF-7凋亡的可能作用机制。方法:采用体外培养人乳腺癌细胞系MCF-7,CCK-8实验检测不同浓度甲基莲心碱对MCF-7细胞增殖抑制作用;乳酸脱氢酶(LDH)试剂盒(微板法)检测细胞上清液LDH含量;流式细胞术分析甲基莲心碱对MCF-7细胞周期及凋亡的影响;实时定量PCR(RT-PCR)检测线粒体凋亡相关基因Bax和Bcl-2的表达水平。结果:CCK-8、LDH结果显示甲基莲心碱以时间、浓度依耐性的方式抑制乳腺癌MCF-7细胞的增殖及促进细胞毒性的增加;流式细胞术结果表明不同甲基莲心碱作用下MCF-7的平均凋亡率分别为(15.44±0.52)、(18.81±2.24)、(24.26±2.84)、(36.90±3.15)、(59.27±5.86),且使其周期阻滞于G0/G1期;RT-PCR检测结果证明甲基莲心碱可上调乳腺癌细胞中促凋亡基因Bax的表达,而下调抑制凋亡基因Bcl-2。结论:甲基莲心碱以时间和浓度依赖的方式抑制乳腺癌细胞增殖、细胞毒性增加,导致细胞周期于G0/G1阻滞并促进癌细胞凋亡。甲基莲心碱抗乳腺癌的可能作用机制是激活线粒体凋亡途径。     

CD47-siRNA通过调控ROS诱导食管癌细胞SEG-1凋亡

目的:探讨CD47-siRNA对食管癌细胞SEG-1凋亡的影响及其机制研究。方法:Western blot法检测食管癌细胞SEG-1中CD47蛋白、促凋亡蛋白Bax和抗凋亡蛋白Bcl-2的表达;CCK-8法检测食管癌细胞SEG-1细胞活力;DCFDA细胞ROS检测试剂盒检测食管癌细胞SEG-1中ROS水平。结果:CD47-siRNA转染组食管癌细胞SEG-1中CD47蛋白明显低于空载对照组(P0.05);CD47-siRNA转染组食管癌细胞SEG-1细胞活力显著低于空载对照组(P0.05);CD47-siRNA转染组食管癌细胞SEG-1中促凋亡蛋白Bax表达显著高于空载对照组(P0.05),抗凋亡蛋白Bcl-2表达低于空载对照组(P0.05);CD47-siRNA转染食管癌细胞SEG-1组ROS水平明显高于空载对照组(P0.05);与CD47-siRNA转染组比较,CAT (ROS抑制剂,5 m M/L, 6 h)预处理增加了细胞活力;与CD47-siRNA转染组比较,CAT (ROS抑制剂,5 m M/L, 6 h)预处理显著降低食管癌细胞SEG-1中Bax蛋白表达以及增加抗凋亡蛋白Bcl-2表达。结论:CD47-siRNA转染通过上调食管癌细胞SEG-1内ROS水平促进食管癌细胞SEG-1凋亡。     

蛇床子素促进人骨肉瘤细胞株SAOS-2凋亡

目的:观察蛇床子素(osthole)对人骨肉瘤细胞SAOS-2增殖和凋亡的影响及潜在的调控机制。方法:采用MTT法、TUNEL染色技术和流式细胞术检测不同浓度蛇床子素对骨肉瘤细胞凋亡的影响;Western blot检测蛇床子素对骨肉瘤细胞中与细胞凋亡密切相关的蛋白(Bax、Bcl-2)的变化。结果:蛇床子素作用于SAOS-2细胞后,MTT结果显示SAOS-2细胞的活力受到明显抑制,且与蛇床子素浓度和时间相关;Western blot结果显示细胞中的促凋亡蛋白Bax表达上调,抗凋亡蛋白Bcl-2表达明显减弱,且呈剂量依赖性。结论:蛇床子素可显著抑制人骨肉瘤细胞的增殖且促进其凋亡的作用,可能与上调凋亡蛋白Bax和下调抗凋亡蛋白Bcl-2的表达有关。     

不同浓度白花丹素对骨肉瘤细胞MG-63凋亡迁移、基质金属蛋白酶及Bcl-2、Bax、Ezrin蛋白表达的影响

目的:研究不同浓度白花丹素对骨肉瘤细胞MG-63凋亡迁移、基质金属蛋白酶(MMP)及Bcl-2、Bax、Ezrin蛋白表达的影响。方法:取对数生长期的骨肉瘤MG-63细胞,传代培养成细胞株后以随机法分成对照组、低剂量组、中剂量组、高剂量组。其中对照组加入到0.1%浓度的DMSO完全培养基中培养,低剂量组、中剂量组、高剂量组分别加入到浓度为5、10、20μmol/L的白花丹素的有关培养基中培养。培养24 h后,采用Transwell法检测MG-63细胞迁移率、Hoechst33342染色法检测MG-63细胞凋亡率、Western blot法检测四组MG-63细胞的MMP-2、MMP-9、Bcl-2、Bax、Ezrin蛋白表达水平。结果:培养24 h后,低剂量组、中剂量组、高剂量组的骨肉瘤细胞MG-63凋亡率及Bax蛋白表达水平均较对照组升高(P<0.05),且随白花丹素浓度的增加而升高(P<0.05);骨肉瘤细胞MG-63的细胞迁移率、MMP-2、MMP-9、Bcl-2及Ezrin蛋白表达水平较对照组降低(P<0.05),且随白花丹素浓度的增加而降低(P<0.05)。结论:白花丹素对骨肉瘤细胞MG-63凋亡的促进作用以及迁移的抑制作用明显,其作用机制可能与抑制骨肉瘤细胞MG-63中的MMP-2、MMP-9、Bcl-2、Ezrin蛋白表达及促进Bax蛋白表达有关,且浓度越高,抑制或促进作用越明显。     

槟榔碱对人乳腺癌MCF-7细胞增殖和凋亡的影响

目的：观察槟榔碱对人乳腺癌细胞（MCF-7）增殖和凋亡的影响,并探讨其机制。方法：采用四甲基偶氮唑盐（MTT）法检测不同浓度（0、10、30、50、100、300、500μmol/L）槟榔碱对MCF-7细胞增殖的影响,Hoechst 33342染色和流式细胞术检测细胞凋亡,Western blot法检测Bax,Bcl-2和P53蛋白表达。结果：低浓度（0、10、30、50μmol/L）槟榔碱不影响细胞的增殖和凋亡;而高浓度（100、300、500 μmol/L）槟榔碱呈浓度依赖性抑制MCF-7细胞增殖、诱导MCF-7细胞凋亡、提高P53和Bax蛋白表达、降低Bcl-2蛋白表达。结论：高浓度槟榔碱抑制MCF-7细胞增殖、诱导凋亡,其机制可能与提高P53和Bax蛋白表达,降低Bcl-2蛋白表达有关。     

不同浓度胆汁酸盐对人肝胆管癌细胞凋亡的影响

目的:探讨不同浓度胆汁酸盐对人肝胆管癌细胞RBE凋亡的影响。方法:采用0、100μM、500μM、1 m M胆汁酸盐作用于人肝胆管癌细胞后,采用流式细胞术检测细胞的凋亡率,Western-blot法检测细胞Bax、Bcl-2蛋白的表达。结果:胆汁酸盐浓度为0、100μM、500μM、1 m M时,人胆管癌细胞的凋亡率分别为(0.7±0.12)%、(0.9±0.15)%、(1.4±0.17)%、(4.8±0.14)%,以1 m M胆汁酸盐所致人胆管癌细胞的凋亡最明显,Bcl-2蛋白表达量逐渐减少,Bax表达量逐渐增多,以1 m M胆汁酸盐时作用最明显,差异具有统计学意义(P0.05)。1 m M胆汁酸盐处理的人肝胆管癌细胞的形态由原来的梭形变成圆形,细胞核固缩、碎裂。结论:胆汁酸盐可以以浓度依赖性的方式导致人胆管癌细胞凋亡,以1 m M胆汁酸盐的作用最明显。     

高浓度葡萄糖对体外培养大鼠胰岛细胞凋亡的影响

目的:探讨高浓度葡萄糖对体外培养大鼠胰岛细胞凋亡的影响及作用机制。方法:SD大鼠胰岛细胞原代培养,不同浓度的葡萄糖处理后,采用MTT比色法检测细胞存活率,Hoechst-PI染色观察细胞凋亡,电镜观察细胞超微结构改变,RT-PCR方法检测Bax及Bcl-2相关基因mRNA表达水平。结果:5.5 mmol·L~(-1),11.1 mmol·L~(-1)和22.2 mmol·L~(-1)葡萄糖处理后,胰岛细胞活性分别下降到78.08%±2.29%、58.39%±3.13%和36.05%±2.63%,与阴性对照组比较差异具有统计学意义(P0.05);Hoechst-PI染色结果显示随着葡萄糖作用浓度的增加,凋亡细胞数量也增加;RT-PCR显示胰岛细胞经不同浓度葡萄糖处理后,Bax mRNA的表达明显上调,Bcl-2 mRNA表达下调(P0.05);电镜观察结果显示随着葡萄糖作用浓度的增加,胰岛细胞超微结构损害程度依次加重。结论:高浓度葡萄糖能明显引起胰岛细胞活性的下降,诱导凋亡反应的发生,凋亡机制与Bcl-2家族蛋白相关。     

华蟾素诱导人胃癌SGC-7901细胞凋亡

本实验旨在探讨华蟾素诱导人胃癌SGC-7901细胞凋亡的作用及其作用机制。采用不同浓度的华蟾素作用胃癌SGC-7901细胞48 h后,MTT法检测细胞活性;光学、荧光显微镜、流式细胞术检测细胞凋亡情况。Real Time RT-PCR和Western Blot分别检测Bax、Bcl-2基因m RNA和蛋白表达水平。结果显示,华蟾素对胃癌SGC-7901细胞增殖具有抑制作用且呈剂量依赖性关系,华蟾素处理7901细胞48 h的IC50值为35.67μg/m L,凋亡率为(5.01±1.69)%。显微镜下观察细胞呈明显凋亡现象,线粒体膜电位(ΔΨm)显著下降(p0.05),细胞阻滞于G1期;Bcl-2的表达下调,Bax的表达明显增加(p0.05,p0.01)。提示华蟾素可能通过上调Bax基因,下调Bcl-2基因诱导人胃癌SGC-7901细胞凋亡。     

降压通络方对大鼠肾小管上皮细胞凋亡的影响

目的:研究降压通络方对大鼠肾小管上皮细胞凋亡的影响。方法:体外培养大鼠肾小管上皮细胞,经过缺血缺氧,将大鼠肾小管细胞随机分为4组:正常组、模型组、降压通络方组、缬沙坦组。采用CCK-8检测细胞增殖情况,免疫组化法检测大鼠肾小管上皮细胞p-AKT蛋白的表达,蛋白免疫印迹法检测大鼠肾小管上皮细胞凋亡相关基因调控蛋白Bcl-2、Bax的表达,对Bcl-2、Bax蛋白的表达水平进行相关性分析。结果:缺血缺氧呈时间依赖性抑制大鼠肾小管上皮细胞活性;免疫组化结果示:p-AKT在正常组呈高表达,模型组低表达,降压通络方组p-AKT表达明显高于模型组(P0.01),缬沙坦组表达低于模型组(P0.05);蛋白免疫印迹法结果示:模型组Bcl-2表达较正常组明显降低(P0.01),降压通络方组和缬沙坦组Bcl-2的表达均高于模型组(P0.05,P0.01);模型组Bax表达较正常组明显升高(P0.01),降压通络方组和缬沙坦组Bax的表达均较模型组降低(P0.05);Bcl-2蛋白与Bax蛋白表达呈负相关性(r=-0.811,P0.01)。结论:降压通络方可抑制大鼠肾小管上皮细胞凋亡,其作用机制可能与上调p-AKT、Bcl-2蛋白,下调Bax蛋白表达有关。     

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.     

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.     

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.     

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.