上皮细胞黏附分子增强细胞间紧密连接促进乳腺癌细胞耐药

乳腺癌是致死率很高的恶性肿瘤,由ABCG2 (ATP-binding cassette G2)介导的多药耐药(multidrug resistance,MDR)是导致其化疗失败的重要原因,探讨ABCG2介导的耐药机制并探寻其关键分子是当前亟待解决的难题。上皮细胞黏附分子(epithelial cell adhesion molecule,EpCAM)参与多种肿瘤耐药,且与乳腺癌MDR密切相关,但它在ABCG2介导的乳腺癌耐药中的作用尚未阐明。本研究目的在于探究EpCAM对于ABCG2介导的乳腺癌细胞的多药耐药的调节作用及其机制。CCK8细胞毒性结果证实,相对于人乳腺癌药物敏感株MCF-7,耐药株MCF-7/MX对米托蒽醌(mitoxantrone,MX)的耐药性显著增强;Western 印迹结果显示,与MCF-7相比,MCF-7/MX细胞中ABCG2高表达,EpCAM表达上调。siRNA法敲低MCF-7/MX细胞中EpCAM可下调其ABCG2表达,并恢复对MX的敏感性。倒置显微镜观察细胞形态,发现敲低EpCAM可减少MCF-7/MX细胞间连接。免疫荧光双染法观察到EpCAM与密封蛋白1(claudin 1)在MCF-7/MX细胞共定位;进一步Western 印迹结果表明,敲低EpCAM减少MCF-7/MX细胞中密封蛋白1表达。综上所述,EpCAM可能通过与密封蛋白1相互作用,增强细胞间紧密连接,促进ABCG2介导的乳腺癌多药耐药。     

P-gp 和MDR1 在三种乳腺癌细胞中表达差异研究

目的:比较P-gp和MDR1在人乳腺癌敏感细胞(MCF-7/S)和耐药细胞(MCF-7/ADR、MCF-7/TAM)中的表达差异,初步探讨乳腺癌细胞对阿霉素与对三苯氧胺产生耐药机制的区别。方法:采用免疫细胞化学法、流式细胞术检测P-gp,采用实时荧光定量PCR法检测MDR1在三种乳腺癌细胞中的表达情况。结果:在MCF-7/ADR细胞中P-gp和MDR1均呈高表达,阳性表达率与MCF-7/S细胞比较,有统计学意义(P<0.01)。在MCF-7/TAM细胞中P-gp、MDR1均呈低表达,与MCF-7/S细胞比较,无统计学意义(P>0.05)。结论:P-gp和MDR1的高表达是乳腺癌细胞对阿霉素产生耐药的主要机制,而并非是乳腺癌细胞对三苯氧胺产生耐药的机制。     

PI3K/AKT 通路参与调控乳腺癌多药耐药和侵袭转移的研究

目的:探讨磷脂酰肌醇-3-激酶/丝苏氨酸蛋白激酶(phosphatidylinositol 3 kinase/serine-threonine kinase,PI3K/AKT)信号通路与乳腺癌多药耐药和侵袭转移的相关性。方法:以乳腺癌细胞系MCF-7为母本,持续低浓度加药诱导建立阿霉素(Adriamycin,ADR)耐药系MCF-7/ADR’。细胞免疫荧光检测两细胞系中磷酸化AKT(phosphorylated AKT,P-AKT)、P-糖蛋白(P-Glycoprotein,P-gp)、基质金属蛋白酶2(matrix metalloproteinase-2,MMP-2)的表达。PI3K抑制剂LY294002作用两系前后,Western Blot检测P-AKT、MMP-2、P-gp的表达改变及qRT-PCR检测MMP-2、MDR1的表达改变。结果:P-AKT、P-gp(MDR1)、MMP-2在MCF-7中为低表达或不表达,MCF-7/ADR’中为高表达。LY294002作用两系后,P-AKT、P-gp(MDR1)、MMP-2在MCF-7/ADR’中的表达明显减低(P<0.05),MCF-7无明显改变。结论:抑制PI3K/AKT信号通路可有效降低MCF-7/ADR’耐药和侵袭转移能力,PI3K/AKT通路是调控乳腺癌多药耐药和侵袭转移的重要信号通路之一。     

HepG2/mdr1稳定细胞系的建立及特性研究

将已构建好的含有人多药耐药（multidrug resistance, MDR）全长基因的真核表达质粒pCI-neo-mdr1,应用脂质体导入人肝癌HepG2细胞,应用G418筛选出人肝癌多药耐药细胞株HepG2/mdr1。通过对HepG2/mdr1细胞形态学的观察和生物学特性的研究,成功地建立了高效、稳定的HepG2/mdr1细胞系;为深入研究肝癌的MDR及其逆转提供了理想的细胞模型,并为探索建立肝癌MDR细胞株提供新的方法和思路,同时为研究肝癌细胞胰岛素抵抗与MDR的关系提供了模型细胞。 将已构建好的含有人多药耐药（multidrug resistance, MDR）全长基因的真核表达质粒pCI-neo-mdr1,应用脂质体导入人肝癌HepG2细胞,应用G418筛选出人肝癌多药耐药细胞株HepG2/mdr1。通过对HepG2/mdr1细胞形态学的观察和生物学特性的研究,成功地建立了高效、稳定的HepG2/mdr1细胞系;为深入研究肝癌的MDR及其逆转提供了理想的细胞模型,并为探索建立肝癌MDR细胞株提供新的方法和思路,同时为研究肝癌细胞胰岛素抵抗与MDR的关系提供了模型细胞。     

两株毛筒腔菌属真菌逆转人乳腺癌细胞多药耐药性

高水平的多药耐药性（multidrug resistance,MDR）基因在肿瘤细胞中过量表达是肿瘤细胞耐药的内在原因,是导致肿瘤化疗失败的主要原素。寻求一种抑制MDR活性的抑制剂是提升抗肿瘤药物药效的重要途径。本研究采用低浓度持续诱导方法建立人乳腺癌细胞（MCF-7）耐药细胞系,结果显示,阿霉素（ADM）、紫杉醇和顺铂对MCF-7耐药细胞系有交叉耐药性,耐药指数（resistance index,RI）分别为5.11、3.55和1.79。菌株对肿瘤细胞的逆转活性筛选表明,红棕毛筒腔菌Tubeufia rubra PF02-2和河池毛筒腔菌T. hechiensis XSL05具有逆转肿瘤细胞多药耐药性为敏感性的活性,逆转倍数（reversion fold,RF）分别为3.79和1.07。结果表明,T. rubra和T. hechiensis具有开发为MDR逆转剂的潜能。     

冬凌草活性部位逆转SGC7901/ADR细胞多药耐药性的体外研究北大核心CSCD

以SGC7901和SGC7901/ADR为细胞模型,检测了冬凌草活性部位与化疗药物联用后,对SGC7901/ADR耐药性的逆转效应;冬凌草活性部位处理细胞后,检测耐药细胞内阿霉素的蓄积变化、耐药细胞P-糖蛋白(P-gp)的表达水平以及mdr1基因的表达变化。结果显示,冬凌草氯仿部位和乙酸乙酯部位可以有效提高化疗药物阿霉素在SGC7901/ADR细胞内的蓄积,降低P-gp的表达,降低mdr1基因的转录。冬凌草逆转胃癌耐药细胞SGC7901/ADR多药耐药性的活性部位是冬凌草氯仿部位和乙酸乙酯部位,其逆转作用与抑制P-gp的表达相关。     

冬凌草活性部位逆转SGC7901/ADR细胞多药耐药性的体外研究

以SGC7901和SGC7901/ADR为细胞模型,检测了冬凌草活性部位与化疗药物联用后,对SGC7901/ADR耐药性的逆转效应;冬凌草活性部位处理细胞后,检测耐药细胞内阿霉素的蓄积变化、耐药细胞P-糖蛋白(P-gp)的表达水平以及mdr1基因的表达变化。结果显示,冬凌草氯仿部位和乙酸乙酯部位可以有效提高化疗药物阿霉素在SGC7901/ADR细胞内的蓄积,降低P-gp的表达,降低mdr1基因的转录。冬凌草逆转胃癌耐药细胞SGC7901/ADR多药耐药性的活性部位是冬凌草氯仿部位和乙酸乙酯部位,其逆转作用与抑制P-gp的表达相关。     

MRTX849逆转ABCB1介导的肿瘤多药耐药

P-糖蛋白(ABCB1/P-gp)过表达是导致肿瘤多药耐药(multidrug resistance, MDR)的主要因素之一。因此,寻找有效的药物来克服ABCB1介导的多药耐药非常重要。MRTX849是一种突变选择性的KRAS G12C共价抑制剂,用于非小细胞肺癌及肠癌的治疗。该实验研究了MRTX849是否能逆转ABCB1介导的多药耐药及其作用机制。逆转实验结果显示, MRTX849能显著逆转ABCB1介导的多药耐药,而不会影响ABCG2介导的多药耐药。Western blot及免疫荧光实验结果显示, MRTX849不会影响ABCB1在细胞内的表达及定位。进一步的研究发现, MRTX849抑制了ABCB1外排功能,导致细胞内药物蓄积水平的增加,从而逆转多药耐药。此外,分子对接实验显示,MRTX849和ABCB1呈现了良好的亲和能力。总之,该研究表明MRTX849逆转了ABCB1介导的多药耐药,为化疗抵抗的肿瘤患者提供了一种新的临床治疗策略。     

沉默AEG-1基因逆转乳腺癌MCF-7/ADM细胞耐药性

本研究旨在分析星形细胞上调基因1(astrocyte elevated gene-1,AEG-1)对乳腺癌MCF-7/ADM细胞化疗药物耐药性的影响,并探讨其作用机制。将MCF-7/ADM细胞在含1.0 mg/L阿霉素(adriamycin,ADM)的培养液中培养以维持细胞的耐药性;应用shRNA技术沉默乳腺癌MCF-7/ADM细胞中AEG-1基因表达;采用MTT比色法检测ADM对MCF-7/ADM的细胞耐毒作用,据以计算ADM的半数抑制浓度(IC50);流式细胞术检测细胞凋亡;Western blot方法检测AEG-1、p53和多药耐药基因1(multidrug resistance gene 1,MDR1)蛋白的表达水平以及Akt、MDM2和Bad的磷酸化水平。结果显示,乳腺癌MCF-7/ADM细胞的AEG-1蛋白水平显著高于MCF-7细胞(P0.05),经shRNA干扰后AEG-1蛋白水平显著降低(P0.05);沉默AEG-1基因能显著降低ADM对MCF-7/ADM细胞的IC50(P0.05),促进MCF-7/ADM细胞凋亡(P0.05),并增强ADM对MCF-7/ADM细胞的促凋亡作用,抑制Akt、MDM2和Bad的磷酸化(P0.05),促进p53蛋白表达(P0.05),降低MDR1蛋白表达水平(P0.05)。结果表明,沉默AEG-1基因可通过促进MCF-7/ADM细胞凋亡和下调MDR1蛋白表达,以逆转MCF-7/ADM细胞对ADM的耐药性。     

肿瘤多药耐药:机制及逆转剂

肿瘤细胞多药耐药性(multidrug resistance,MDR)的产生是临床上导致肿瘤化疗失败的主要原因之一,因此寻找高效低毒的MDR逆转剂已成为肿瘤药物开发领域的热点。MDR的作用机制主要包括P-糖蛋白、多药耐药相关蛋白、乳腺癌耐药蛋白、肺耐药相关蛋白等等。多药耐药逆转剂包括钙离子通道阻滞剂、维拉帕米及其衍生物等等。本文主要介绍了MDR的作用机制以及肿瘤多药耐药逆转剂的研究进展。     

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.     

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.     

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.     

Chiral Separation of G‐type Chemical Warfare Nerve Agents via Analytical Supercritical Fluid Chromatography

Chemical warfare nerve agents (CWNAs) are extremely toxic organophosphorus compounds that contain a chiral phosphorus center. Undirected synthesis of G‐type CWNAs produces stereoisomers of tabun, sarin, soman, and cyclosarin (GA, GB, GD, and GF, respectively). Analytical‐scale methods were developed using a supercritical fluid chromatography (SFC) system in tandem with a mass spectrometer for the separation, quantitation, and isolation of individual stereoisomers of GA, GB, GD, and GF. Screening various chiral stationary phases (CSPs) for the capacity to provide full baseline separation of the CWNAs revealed that a Regis WhelkO1 (SS) column was capable of separating the enantiomers of GA, GB, and GF, with elution of the P(+) enantiomer preceding elution of the corresponding P(–) enantiomer; two WhelkO1 (SS) columns had to be connected in series to achieve complete baseline resolution. The four diastereomers of GD were also resolved using two tandem WhelkO1 (SS) columns, with complete baseline separation of the two P(+) epimers. A single WhelkO1 (RR) column with inverse stereochemistry resulted in baseline separation of the GD P(–) epimers. The analytical methods described can be scaled to allow isolation of individual stereoisomers to assist in screening and development of countermeasures to organophosphorus nerve agents. Chirality 26:817–824, 2014. © 2014 The Authors. Chirality published by John Wiley Periodicals, Inc.     

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.     

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.     

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.     

八种脑-肠肽侧脑室内注射对大鼠基础胃酸分泌的影响

用乌拉坦麻醉大鼠作急性实验,采用连续灌流胃并收集流出液的方法,观察向侧脑室内注射微量脑-肠肽对大鼠基础胃酸分泌的影响。实验结果如下:(1)雨蛙肽、八肽胆囊收缩素、促甲状腺素释放激素及四肽胃泌素均使总酸排出量增加;(2)生长抑素、胰多肽、P 物质、胰高血糖素则使总酸排出量减少;(3)上述肽类用侧脑室注射的剂量作肌肉注射,除四肽胃泌素也产生明显的刺激胃酸分泌作用外,对胃酸分泌均无明显影响。以上结果提示,脑内的一些肽类可能以神经递质或调制物的方式,参与中枢对胃酸分泌的调节。     

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.     

A chromium-tolerant plant growing in Cr-contaminated land

In this paper, a kind of Typhaceae plant species, Typha angustifolia L, with a high tolerance to Cr is described. Experiments were carried out to examine its ability to tolerant Cr and its physiological response. The results showed that there was no difference in growth, plant height, and biomass response to external Cr (VI) between the plants exposed to 100 microM Cr (VI) and control (0 microM), while increasing Cr levels to 200-800 microM induced a significant decrease in plant height and biomass, but no significant injury was detected, even for the plants exposed to 800 microM Cr. Chromium induced significant increases in superoxide dismutase (SOD) and peroxidase (POD) activities. Meanwhile, a significantly positive correlation was found between Cr and Mn or Cu in leaves and roots, respectively. The Cr tolerance of the plant appeared to be associated with the enhancement of SOD and POD activities and the improvement in uptake and translocation of the essential microelements.