TRAIL与顺铂联用对人骨肉瘤细胞杀伤作用的实验研究

目的探讨TRAIL与顺铂联合应用对人骨肉瘤细胞的杀伤作用.方法将TRAIL与顺铂单独及联合应用于体外培养的骨肉瘤OS-732细胞,采用MTT法测定细胞抑制率,于倒置相差显微镜、荧光显微镜、扫描及透射电镜下观察细胞的形态学改变及超微结构变化.结果 50ng/ml的TRAIL与5μg/ml顺铂合用于OS-732细胞24小时后,细胞抑制率为84.37%,明显高于单用TRAIL(50μg/ml)时的9.68%及单用顺铂(5μg/ml)时的24.39%(P<0.01).细胞的形态与超微结构观察均显示,合用比单用促凋亡效应更显著.结论 TRAIL与顺铂合用,可高效杀伤人骨肉瘤细胞,此协同作用主要是通过诱导肿瘤细胞凋亡来实现的.     

川楝素诱导人肺癌A549细胞凋亡

该研究旨在探讨川楝素诱导人肺癌A549细胞凋亡作用及其作用机制。通过不同浓度的川楝素作用于A549细胞48 h后,采用MTT法检测细胞活性;光学显微镜及荧光显微镜下观察细胞形态结构;流式细胞术检测细胞凋亡率、线粒体膜电位(ΔΨm)和细胞周期;实时定量RTPCR和Western blot分别检测Bax、Bcl-2、Fas、Cycs(细胞色素C)和Caspase-3基因m RNA和蛋白质水平。结果显示,在一定浓度范围内,川楝素能抑制A549细胞增殖,诱导细胞凋亡,且呈剂量依赖性。川楝素作用48 h的最佳药物浓度是40μmol/L,增殖抑制率为46.73%±1.47%,细胞凋亡率为13.18%±0.41%,线粒体膜电位(ΔΨm)显著下降(P0.01),细胞阻滞于G2期和S期;Bcl-2的表达显著降低,Bax、Fas、Cycs和Caspase-3的表达显著增加(P0.01),提示川楝素可能通过上调Bax、Fas、Cycs和Caspase-3基因和下调Bcl-2基因诱导人肺癌A549细胞凋亡。     

褪黑素抑制人骨肉瘤U2细胞增殖及其机制

目的研究褪黑素对体外培养人骨肉瘤U2细胞增殖的影响,并对其可能机制进行研究。方法体外培养U2细胞,用不同浓度褪黑素加以干预,以CCK-8法检测褪黑素对U2细胞增殖的影响;对细胞进行Hoechst 33258染色,荧光显微镜下观察褪黑素对U2细胞凋亡的影响;以流式细胞术检测褪黑素对细胞凋亡及细胞周期的影响;通过实时荧光定量PCR及免疫印迹法检测U2细胞Fas基因表达情况。结果 1 CCK8法检测显示褪黑素对U2细胞增殖有抑制作用,作用时间越长,浓度越高,抑制作用越明显;2 Hoechst33258染色可见凋亡小体;3流式细胞检测显示褪黑素作用后早期凋亡的U2细胞增加,并使细胞周期阻滞在G2/M期;4实时荧光定量PCR及免疫印迹法显示褪黑素处理后U2细胞上调Fas蛋白表达。结论褪黑素对U2细胞的增殖有抑制作用,可诱导细胞发生凋亡,其机制可能与将细胞复制周期阻滞在G2/M期及上调Fas蛋白表达量有关。     

姜黄素对人成骨肉瘤MG-63细胞增殖抑制和凋亡相关基因表达的影响

目的:探讨姜黄素对人成骨肉瘤MG-63细胞增殖抑制及凋亡相关基因表达的影响.方法:用姜黄素处理MG-63细胞,细胞计数法检测细胞增殖抑制的效果;荧光染色观察细胞的凋亡;流式细胞仪(FCM)进行细胞周期时相分析;免疫细胞化学法和western blotting免疫法检测细胞凋亡相关基因的表达水平.结果:随着姜黄素浓度的增加及其作用时间延长,对细胞的增殖抑制作用增强,最高抑制率可达89.07%;光镜下可观察到细胞发生染色质浓缩,细胞核凝聚和碎裂等典型的凋亡形态学改变;FCM检测结果显示细胞经姜黄素处理后出现明显的凋亡峰;免疫反应结果显示,凋亡相关基因bcl-2和P53表达水平降低,而bax和Fas表达水平升高.结论:姜黄素能显著抑制MG-63细胞增殖并可有效诱导其凋亡,其作用机制可能与bcl-2和bax二者的比值发生变化从而接受了凋亡刺激信号有关.     

Desmosdumotin-C A环衍生物TEP诱导HL-60细胞凋亡作用机制研究

本研究主要探讨毛叶假鹰爪素C(Desmosdumotin C)A环衍生物TEP诱导人急性白血病HL-60细胞凋亡作用及机制。流式细胞技术检测TEP诱导细胞凋亡及其对凋亡细胞中Fas、Fas L、Bax、Bcl-2表达率的影响,透射电镜观察凋亡细胞形态学改变。结果显示40μg/m L TEP作用细胞24 h后,细胞可呈现典型的凋亡形态学变化;40μg/m L TEP可明显提高凋亡细胞中Fas、Fas L、Bax的表达(P0.05),并可明显降低抗凋亡细胞Bcl-2的表达(P0.05)。以上实验结果表明毛叶假鹰爪素C(Desmosdumotin C,Des C)A环衍生物TEP可有效地诱导HL-60细胞凋亡,其作用机制可能与上调Fas、Fas L、Bax表达以及下调Bcl-2表达有关。     

莪术油诱导人胃腺癌SGC-7901细胞凋亡的研究

该实验目的是探究莪术油(zedoray turmeric oil,ZTO)诱导人胃腺癌SGC-7901细胞凋亡的作用。不同浓度的ZTO作用人胃腺癌SGC-7901细胞48 h后,采用台盼兰拒染法检测细胞生长抑制率;光学显微镜、荧光显微镜、透射电镜下观察细胞的形态和超微结构;琼脂糖凝胶电泳检测DNA片段化;Annexin V-FITC法检测细胞的凋亡率;实时荧光定量PCR和Western blot检测Bax、Bcl-2 m RNA和蛋白表达水平。结果显示,作用人胃腺癌SGC-7901细胞48 h,ZTO的最佳浓度是110μg/m L,IC50为(108.002±0.305)μg/m L;显微镜下细胞呈不同程度的凋亡特征;DNA电泳可见梯状条带;最佳药物浓度作用细胞48 h的早期凋亡率为(25.07±0.82)%;Bax表达水平升高,Bcl-2表达水平降低,Bax/Bcl-2比值显著升高(P0.05),表明ZTO通过上调Bax表达、下调Bcl-2表达诱导人胃腺癌SGC-7901细胞凋亡。     

重组荞麦胰蛋白酶抑制剂对人肝癌细胞的凋亡及半胱氨酸天冬酶活性的影响

先前的研究表明,基因重组荞麦胰蛋白酶抑制剂 (rBTI) 具有诱导不同肿瘤细胞凋亡的作用.为了揭示其诱导肿瘤细胞凋亡的可能机理,从基因水平上探讨与凋亡有关的分子事件,本研究用不同浓度的 rBTI 体外作用于人肝癌细胞 HepG2 后,采用 MTT 比色法检测抑制剂对epG2 细胞的抑制率,用 DNA 凝胶电泳和细胞核的形态学观察检测 HepG2 细胞的凋亡.结果表明,rBTI 在体外能够明显抑制 HepG2 细胞的增长,并诱导细胞凋亡.另外,细胞凋亡与Bcl-2/Bax mRNA 水平有关.通过 RT-PCR 检测发现,细胞经过rBTI处理后,抗凋亡基因Bcl-2 mRNA 水平下调,促凋亡基因 Bax mRNA 有所上调,而对照 GAPDH 无变化.对 HepG2细胞中 Fas/Fas 配体及半胱氨酸天冬酶（caspase）的研究证明,细胞经过 rBTI 处理后,对死亡受体 Fas mRNA没有影响; rBTI 可明显激活caspase-3 和 caspase-9 酶活性, 对caspase-8 活性几乎无影响.上述结果表明,rBTI 对HepG2 细胞具有明显的诱导凋亡作用,其诱导细胞凋亡的机制与 caspase-3 依赖性凋亡调节信号通路有关,未涉及 Fas/Fas 配体途径.     

维甲酸对人成骨肉瘤MG-63细胞增殖和相关基因表达的影响

目的:研究维甲酸对人成骨肉瘤MG-63细胞增殖和相关基因表达的影响,以探索其对成骨肉瘤细胞的生物学效应.方法:以1μmol/L维甲酸处理人成骨肉瘤MG-63细胞,生长曲线测定,流式细胞仪分析、光镜观察和免疫细胞化学检测等研究维甲酸对MG-63细胞的生长曲线、细胞周期和相关癌基因、抑癌基因表达的影响,并对其作用机理进行初步分析.结果:维甲酸处理7天后,MG-63细胞生长抑制率达到42.2%,G0/G1期比例达到61.8%,细胞形态铺展,排列趋于规则,癌基因c-myc、c-fos的表达降低.而抑癌基因Rb、p27表达上调.结论:1μmol/L维甲酸可以有效抑制细胞的增殖活动,改变细胞恶性形态特征,下调癌基因c-myc、c-fos和上调抑癌基因Rb、p27的表达,从而对人成骨肉瘤细胞分化具有诱导作用.     

苦参碱对体外人髓母细胞瘤D341细胞凋亡、增殖及凋亡相关蛋白的影响

目的:探讨苦参碱在体外对人髓母细胞瘤D341细胞的凋亡、增殖作用及相关蛋白Caspase 3、Caspase 9表达的影响。方法:D341细胞体外成功培养后,按所加苦参碱的浓度进行分组(0.5 mg/ml、1.0 mg/ml、1.5 mg/ml、2.0 mg/ml,0 mg/ml为对照组),各实验重复3次。光学显微镜、透射电镜观察细胞形态、结构的改变,CCK-8法检测D341细胞增殖的影响,Annexin-V FITC/PI双染法检测细胞凋亡。Western blot检测苦参碱作用D341细胞后Caspase 3、Caspase 9蛋白表达水平的改变。结果:在苦参碱作用下,细胞增殖的抑制率随药物浓度的增加而逐渐升高,抑制率有统计学差别(P0.01);药物作用时间不同对细胞增殖的抑制程度不同,24 h与72 h组的抑制率有统计学差别(P0.01)。瘤细胞的凋亡率随着药物浓度的增加而逐渐升高,0.5 mg/ml、1.0 mg/ml与1.5 mg/ml、2.0mg/ml组间的差别均有统计学意义(P0.05);瘤细胞的凋亡率随着作用时间延长而升高,24 h与72 h、48 h与72 h组间的凋亡率的差别有统计学意义(P0.05)。随着苦参碱浓度的增加,细胞Caspase 3和Caspase 9的表达均逐渐增强(P0.01)。结论:苦参碱在体外对D341细胞是通过上调Caspase 3和Caspase 9蛋白来发挥其促凋亡、抑制增殖作用。     

EGFR信号通路在SiO2诱导肺上皮细胞间质转化中的作用机制

目的：在二氧化硅（SiO2）刺激下可引起肺部一系列的炎症反应及其伴随相关的成纤维细胞增殖,然而EGFR信号通路可维持细胞增殖、分化和凋亡的平衡,因此,我们可以设想EGFR信号通路是否在肺纤维化的发生发展中起到重要的作用。本实验探讨SiO2是否能诱导人肺上皮细胞（A549）发生上皮间质转化,并且研究EGFR信号通路在矽肺纤维化中的作用机制。方法：以A549为研究对象,用0（对照组）、50、100、200μg／mlSiO2孵育A549,作用48h后于倒置显微镜观察细胞形态学改变,并收集不同时段细胞,采用实时荧光定量PCR（RT-PCR）检测E-钙黏蛋白（E-cadherin）和α-平滑肌肌动蛋白（α-SMA）mRNA表达变化,细胞免疫荧光方法检测E-cadllerin、α-SMA及信号转导蛋白EGFR表达的变化。结果：倒置显微镜观察A549经SiO2处理后细胞形态由鹅卵石状转变为纺锤型或梭型,形态似成纤维细胞,随着SiO2浓度的升高,E-cadmRNA和蛋白表达逐渐下调,在200μg／ml组表达最低,α-SMAmRNA和蛋白表达逐渐上调,200μg／ml组α-SMA表达最高;EGFR蛋白表达上调;50、100、200μg／ml与对照组的差异具有统计学学意义（P〈0．05）。结论：SiO2可诱导肺上皮细胞向间质细胞转化,其机制可能与EGFR信号通路有关。     

Land,livestock, and livelihoods: Changing dynamics of gender,caste, and ethnicity in a Nepalese Village

Over the past 10 years, Ghusel VDC, Lalitpur District has moved from primarily subsistence agriculture into the wider cash economy aided by the Small Farmers' Development Program (SFDP), which provides credit to farmers mainly for the purchase of buffalo for milk production, and by the National Dairy Corporation, which supports local dairy cooperatives. Analysis reveals that buffalo-keeping and milk sales are increasing the well-being of many households, while at the same time creating new inequalities in gender roles and responsibilities, greater inequities between Brahmin and Tamang residents in Ghusel, and placing pressures on the ecosystem for increased supplies of fodder and fuelwood. Evidence suggests that there is critical, need for attention to the social, and particularly gender-based, implications of maintaining livestock for milk sales and to the ecological underpinnings of this livelihood system.     

Reorientation of Microfibrils and Microtubules at the Outer Epidermal Wall of Maize Coleoptiles During Auxin-Mediated Growth

Auxin-mediated elongation growth of isolated subapical coleoptile segments of maize (Zea mays L.) is controlled by the extensibility of the outer cell wall of the outer epidermis (Kutschera et al., 1987). Here we investigate the hypothesis that auxin controls the extensibility of this wall by changing the orientation of newly deposited microfibrils through a corresponding change in the orientation of cortical microtubules. On the basis of electron micrographs it is shown that cessation of growth after removal of the endogenous source of auxin is correlated with a relative increase of longitudinally orientated microfibrils and microtubules at the inner wall surface. Conversely, reinduction of growth by exogenous auxin is correlated with a relative increase of transversely orientated microfibrils and microtubules at the inner wall surface. These changes can be detected 30–60 min after the removal and addition of auxin, respectively. The functional significance of directional changes of newly desposited wall microfibrils for the control of elongation growth is discussed.     

多马胺能药物对鲇鱼促性腺激素（GtH）分泌活动的影响

以珠江流域鲇鱼（silurus asotus)为实验材料,研究了多巴胺（DA）能药物（DA及其D－2型受体拮抗物 ,DOM）对鲇鱼促性腺激素（GtH)释放的影响,结果表明,在性腺发育的各个时期,单独注射DOM（5ug/g)均不能显著提高鲇鱼血液基础GtH水平,当DOM与LHRH－A联合注射时能显著增强LHRH－A刺激GtH释放的作用;DA只能抑制GnRH诱导的GtH释放,对基础GtH释放无抑制作用,这种生殖内分泌调节方式与鲇形目的革胡子鲇（Clarias gariepinus)和大鳍Hu（Mystus macropterus)相似,而与鲤形目的鲁科（Cyrpindiae)鱼类不同。     

Influence of Fluoride on Rat Kidney Antioxidant System: Effects of Methionine and Vitamin E

The aim of the study has been to determine and compare the influence upon the kidney antioxidative system, exercised by administration of vitamin E, and vitamin E in combination with methionine, under conditions of oxidative stress induced by sodium fluoride. The experiment was carried out on Wistar FL rats (adult males) that, for 35 days, were administered water, NaF, NaF with vitamin E, or vitamin E with methionine (doses: 10 mg NaF/kg of body mass/24 h, 3 mg vitamin E per 10 μl per rat for 24 h, 2 mg methionine per rat for 24 h). The influence of administered sodium fluoride and antioxidants upon the antioxidative system in kidney was examined by analyzing the concentration of malondialdehyde (MDA) and the activity of the most important antioxidative enzymes (SOD, total and both its isoenzymes, GPX, GST, GR, and CAT). The studies carried out confirmed the disadvantageous effect of the administered dose of NaF upon the antixodiative system in rats (increase in the concentration MDA, decrease activity of all antioxidative enzymes). The administration of vitamin E increased the activity of studied enzymes with the exception of glutathione reductase GR; it also reduced the procesess of lipid peroxidation. It has been found that combined doses of vitamin E and methionine were most effective in inhibiting lipid peroxidation processes. The results confirmed the antioxidative properties of methionine.     

Gamma-glutamylcysteine protects ergothioneine-deficient Mycobacterium tuberculosis mutants against oxidative and nitrosative stress

Mycobacterium tuberculosis (M.tb.), the causative agent of tuberculosis (TB), cannot synthesize GSH, but synthesizes two major low molecular weight thiols namely mycothiol (MSH) and ergothioneine (ERG). Gamma-glutamylcysteine (GGC), an intermediate in GSH synthesis, has been implicated in the protection of lactic acid bacteria from oxidative stress in the absence of GSH. In mycobacteria, GGC is an intermediate in ERG biosynthesis, and its formation is catalysed by EgtA (GshA). GGC is subsequently used by EgtB in the formation of hercynine-sulphoxide-GGC. In this study, M.tb. mutants harbouring unmarked, in-frame deletions in each of the fives genes involved in ERG biosynthesis (egtA, egtB, egtC, egtD and egtE) or a marked deletion of the mshA gene (required for MSH biosynthesis) were generated. Liquid chromatography tandem mass spectrometry analyses (LC-MS) revealed that the production of GGC was elevated in the MSH-deficient and the ERG-deficient mutants. The ERG-deficient ΔegtB mutant which accumulated GGC was more resistant to oxidative and nitrosative stress than the ERG-deficient, GGC-deficient ΔegtA mutant. This implicates GGC in the detoxification of reactive oxygen and nitrogen species in M.tb.     

Tuning of electronic properties of one-dimensional cyano-bridged Cu-Ni, Cu-Pd, and Cu-Pt bimetallic assemblies by stereochemistry of ligands

Preparations, XPS and electronic spectroscopy, and magnetism of seven new one-dimensional cyano-bridged coordination polymers, chiral [Cu(RR-chxn)₂][Pd(CN)₄] · 2H₂O (1), [Cu(trans-chxn)₂][M(CN)₄] · 2H₂O (2, 4, and 6 for M = Pd, Ni, and Pt), and [Cu(cis-chxn)₂][M(CN)₄] · 2H₂O (3, 5, and 7 for M = Pd, Ni, and Pt) (RR-chxn = cyclohexane-(1R,2R)-diamine, trans-chxn = racemic trans-cyclohexane-(1,2)-diamine, and cis-chxn = racemic cis-cyclohexane-(1,2)-diamine) have been reported in view of tuning of their electronic properties by stereochemistry of chxn ligands and metal-substitution. Comparison of Cu 2p_1/2 and 2p_3/2 peaks of XPS and broad d-d bands around 18 000 cm⁻¹ of electronic spectra are described systematically for 1-7. Variable-temperature magnetic measurement shows that complexes 1-7 indicate weak antiferromagnetic interactions via cyano-bridges. Because of semi-coordination coupled with pseudo Jahn-Teller elongation and electrostatic interaction for 1, the axial Cu-N coordination bond distances of 2.330(7) and 3.092(8) Å are considerably longer than those of equatorial ones in the range from 2.016(6) to 2.030(6) Å. The former bond distances of 1 are intermediate values among the related Ni (2.324(6) and 3.120(8) Å) and Pt (2.34(1) and 3.09(1) Å) complexes.     

Identification of specific binding sites for leukotriene C4 in human fetal lung

Specific leukotriene C₄ (LTC₄) binding sites were identified in membrane preparations from human fetal lung. Specific binding of [³H]-LTC₄ represented 95 percent of total binding, reached steadystate within 10 minutes and was rapidly reversible upon addition of excess unlabeled LTC₄. Binding assays were performed at 4°C under conditions which prevented metabolism of [³H]-LTC₄ (80 mM serineborate, 10 mM cysteine, 10 mM glycine). Under these conditions, greater than 95 percent of the membrane bound radioactivity, as analyzed by high performance liquid chromatography, co-eluted with the LTC₄ standard. Computer-assisted analyses of saturation binding data showed a single class of binding sites with a dissociation constant (K_d) of 26 + 6 nM and a density (B_max) of 84 ± 18 pmol/mg protein. Pharmacological specificity was demonstrated by competition studies in which specific binding of [³H]-LTC₄ was displaced by LTC₄ and its structural analogs with inhibition constants (K_j) of 10 to 30 nM, whereas LTD₄, diastereoisomers of LTD₁, LTE₄ and the end organ antagonist FPL 55712 were 150 to 700 fold less potent competitors than LTC₄. These results provide evidence for specific, reversible, saturable, high affinity binding sites for [³H]-LTC₄ in human fetal lung membranes.     

Structure and function of S-adenosylhomocysteine hydrolase

In mammals, S-adenosylhomocysteine hydrolase (AdoHcyase) is the only known enzyme to catalyze the breakdown of S-adenosylhomocysteine (AdoHcy) to homocysteine and adenosine. AdoHcy is the product of all adenosylmethionine (AdoMet)-dependent biological transmethylations. These reactions have a wide range of products, and are common in all facets of biometabolism. As a product inhibitor, elevated levels of AdoHcy suppress AdoMet-dependent transmethylations. Thus, AdoHcyase is a regulator of biological transmethylation in general. The three-dimensional structure of AdoHcyase complexed with reduced nicotinamide adenine dinucleotide phosphate (NADH) and the inhibitor (1′R, 2′S, 3′R)-9-(2′,3′-dihyroxycyclopenten-1-yl)adenine (DHCeA) was solved by a combination of the crystallographic direct methods program, SnB, to determine the selenium atom substructure and by treating the multiwavelength anomalous diffraction data as a special case of multiple isomorphous replacement. The enzyme architecture resembles that observed for NAD-dependent dehydrogenases, with the catalytic domain and the cofactor binding domain each containing a modified Rossmann fold. The two domains form a deep active site cleft containing the cofactor and bound inhibitor molecule. A comparison of the inhibitor complex of the human enzyme and the structure of the rat enzyme, solved without inhibitor, suggests that a 17° rigid body movement of the catalytic domain occurs upon inhibitor/substrate binding.     

In vitro studies of the rabbit immune system. IV. Differential mitogen responses of isolated T and B cells.

The mitogenic responses of separated rabbit lymphocyte populations functionally analogous to mouse T and B cells have been tested in vitro. Purified T cells were prepared by passage over nylon wool (NW) and purified B cells prepared by treatment with antithymocyte serum and complement (ATS + C). ATS + C kills 70% of peripheral blood lymphocytes (PBL's) and 50% of the spleen cells while passage over NW yields 40% of the applied PBL's and 5–23% of the applied spleen cells. NW-purified T cells from the spleen or PBL's respond fully to concanavalin A (Con A) but have a reduced response to phytohemaglutinin (PHA) and little or no response to goat anti-rabbit immunoglobulin (anti-Ig). PBL's that survive ATS + C (B cells) are stimulated by anti-Ig but not by Con A or PHA. B cells purified from spleen do not respond to Con A or PHA but will respond to anti-Ig under appropriate conditions. A full spleen B-cell response to anti-Ig required removal of Ig produced by the cultures that blocked anti-Ig stimulation. It is concluded that, for rabbit lymphocytes, Con A and PHA are primarily T-cell mitogens and that anti-Ig is primarily a B-cell mitogen. However, the mitogen response of unfractionated PBL or spleen cell populations indicates an overlap in reactivity. This could be due to cells sharing T and B properties, alteration of cell populations by the fractionation procedures used, or recruitment of one population in the presence of a mitogenic response of the other population.     

Cellulose-microfibril-orienting mechanisms in plant cells walls

A brief review is given of the changing views over the years, as knowledge of wall structure has developed, concerning the mechanism whereby cellulose chains may be oriented. This leads to an examination of current concepts, particularly those concerning microtubules. It is shown that none of the mechanisms suggested whereby microtubules might cause orientation of cellulose microfibrils is consistent with the known range of molecular architectures found in plant cell walls. It is further concluded that any mechanism which necessitates an indissoluble link between the plasmalemma and the cellulose-synthesising complex at the tip of a microfibril is unacceptable. A new proposal is presented in which it is speculated that both microtubules and microfibrils are oriented by a mechanism separate from both. It is shown that if two vectors are contemplated, one parallel to cell length and one at right angles, and a sensor exists on the plasmalemma surface which responds to changes in the vectors, then all known wall structures may be explained. The possible nature of the vectors and the sensor are considered.