人EGFR显性负性突变体抑制胃癌细胞促血管形成能力

目的探讨人表皮生长因子显性负性突变体(dominant negative epidermal growth factor receptor,DNEGFR))对胃癌细胞促血管形成能力的影响及其分子机制,并检测其对裸鼠皮下移植瘤生长的影响。方法选用2株人胃癌细胞,分为如下6组:SGC-7901及NCI-N87细胞未转染组(US组,UN组),SGC-7901及NCI-N87细胞pEGFP-N1质粒转染组(ES组,EN组),SGC-7901及NCI-N87细胞pEGFPN1-DNEGFR质粒转染组(DS组,DN组)。采用人脐静脉内皮细胞(humanumbilical vein endothelial cell,HUVEC)管腔结构形成实验检测体外促血管形成能力,采用酶联免疫吸附测定法(enzyme-linked immunosorbent assay,ELISA)测定细胞培养液中血管内皮生长因子(vascular endothelial growth factor,VEGF)的水平,建立人胃癌细胞裸鼠移植瘤模型,标本微血管密度(microvessel density,MVD)检测体内促血管形成能力,标本体积检测其对裸鼠皮下移植瘤生长的影响。结果转染pEGFPN1-DNEGFR质粒的人胃癌细胞株出现HUVEC管腔结构形成抑制,培养液中VEGF水平降低,MVD计数降低,裸鼠皮下移植瘤体积变小。结论 DNEGFR可能通过下调VEGF分泌抑制胃癌细胞体外及裸鼠体内促血管形成能力,最终抑制裸鼠皮下移植瘤生长。     

人β防御素-2通过TGF-β/Smad信号通路对胃癌SGC7901细胞增殖、迁移和侵袭的影响

该文探讨了人β防御素-2(hBD2)对胃癌SGC7901细胞的增殖、迁移和侵袭的影响。将真核表达载体pCMV-hBD2转染于人胃癌SGC7901细胞。采用qPCR和免疫印迹法(Western blot)检测转染效率。Western blot检测TGF-β1、p-Smad2/3、Smad2/3、MMP9的蛋白表达水平。Transwell法检测SGC7901细胞迁移和侵袭能力。EdU法和流式细胞术分别检测增殖能力与细胞周期。结果显示,转染真核表达载体pCMV-hBD2的SGC7901细胞, hBD2的表达水平明显高于SGC7901和转染pCMV-Blank的SGC7901细胞。SGC7901-hBD2细胞中TGF-β1、p-Smad2/3和MMP9的蛋白表达水平均低于SGC7901和SGC7901-Blank细胞,而Smad2/3表达水平不变。同时,其迁移侵袭和增殖能力均受到抑制,细胞周期G0/G1期阻滞。该实验结果表明, hBD2可能通过下调TGF-β/Smad信号通路调控SGC7901细胞的迁移侵袭以及增殖能力。     

RNA干扰NRP2基因表达对胃癌细胞SGC7901裸鼠移植瘤的抑制作用

目的:探讨特异性抑制NRP2基因表达对人胃癌细胞SGC7901裸鼠移植瘤生长及淋巴管新生的影响。方法:采用小RNA干扰方法,构建shNRP2质粒,稳定转染入SGC7901细胞株,Westen blot检测转染前后NRP2蛋白的表达。建立人胃癌细胞SGC7901裸鼠移植瘤模型,随机分为shNRP2组(实验组)、shCon组(HK阴性对照组)和正常对照组,观察移植瘤的生长情况。6周后处死裸鼠,免疫组化检测NRP2蛋白的表达及微淋巴管密度(Micro-vessel density,MLD)。结果:成功构建shNRP2质粒,与另两组比较,shNRP2组细胞NRP2蛋白表达明显降低,且移植瘤组织生长、NRP2表达及MLD明显受抑制。结论:抑制NRP2基因的表达,可以抑制胃癌裸鼠移植瘤的生长及淋巴管的形成,NRP2基因有可能成为一个潜在的胃癌生物治疗靶点。     

白黎芦醇对胃癌SGC 一7 901 细胞V EGF 表达的影响

目的：探讨白藜芦醇（resveratrol,Res）在体外对胃癌SGC-7901细胞VEGF表达的影响。方法：体外培养胃癌SGC-7901细胞,MTT法检测白藜芦醇对SGC-7901细胞的增殖抑制作用,RT—PCR方法检测VEGFmRNA表达,免疫细胞化学检测VEGF蛋白的表达。结果：白藜芦醇呈时间剂量性抑制胃癌细胞SGC7901的增殖;胃癌SGC-7901细胞高水平表达VEGF,白藜芦醇能显著降低胃癌SGC-7901细胞VEGFmRNA和蛋白表达。结论：白藜芦醇可以下调胃癌SGC-7901细胞VEGF的表达,抑制胃癌细胞的增殖。     

Ndrg2基因表达对胃癌细胞增殖调控及其机理的研究

为研究Ndrg2基因在人类肿瘤发生发展中的作用,以不表达Ndrg2基因的胃癌细胞系HGC-27和表达Ndrg2基因的胃癌细胞系SGC-7901作为对比材料,以Ndrg2基因转染HGC-27胃癌细胞系,以及用Ndrg2的反义寡核苷酸封闭SGC-7901胃癌细胞系中Ndrg2基因的表达.发现Ndrg2可以抑制HGC-27胃癌细胞的软琼脂集落形成,有一定诱导细胞凋亡的作用,对细胞周期蛋白E的表达有明显下调作用.当封闭了SGC-7901胃癌细胞中Ndrg2基因表达的软琼脂集落形成受到抑制,流式细胞仪检测发现此时的SGC-7901细胞周期被阻滞在G1期,细胞周期蛋白D1和E表达下调.Ndrg2基因对两种肿瘤细胞中的细胞外信号调节激酶(ERK)和P38的表达也有不同的影响.     

PDSW联合热疗对SGC-7901细胞PTEN和Ki67基因及蛋白表达的影响

为探讨生理性深层海水(PDSW)联合热疗对人胃癌SGC-7901细胞体外和体内的抗癌作用,通过在体外培养人胃癌SGC-7901细胞株,随机分为实验组、对照组和空白组。实验组加PDSW,对照组加生理盐水,空白组不做其他处理。分别在37℃、40℃和43℃热疗温度下培养。同时,将SGC-7901细胞注射至裸鼠皮下,建立移植瘤模型,随机分为5组,至肿瘤长至直径为1 cm时,分别进行H_2O+RT、H_2O+40℃、H_2O+43℃、PDSW+40℃、PDSW+43℃处理,用实时荧光定量PCR检测细胞和组织中PTEN和Ki67基因表达情况,Western Blot检测PTEN和Ki67蛋白的表达情况。结果发现,PDSW联合热疗处理后,SGC-7901细胞和裸鼠皮下移植瘤组织中PTEN基因和蛋白的表达量上升幅度较大,而Ki67基因和蛋白的表达量较低,说明PDSW联合热疗能激活胃癌SGC-7901细胞中PTEN基因和蛋白上调表达,而抑制Ki67基因和蛋白的表达,从而有效抑制胃癌细胞的增殖。     

RNA干扰抑制Snail表达对于胃癌细胞上皮间充质转化以及体外侵袭的影响

目的用RNA干扰(RNAinterference,RNAi)技术抑制转录因子Snail表达,观察其对人胃腺癌SGC-7901细胞上皮-间充质转化表型和体外侵袭能力的影响。方法构建能表达针对Snail的小干扰RNA(Small interferingRNA,si RNA)的RNA干扰载体(Snail si RNAvector)和表达不针对任何已知mRNA的si RNA的阴性对照RNA干扰载体(control si RNAvector),分别转染SGC-7901细胞,筛选得到Snail表达受抑制的SGC-7901-siSnail细胞和Snail表达未受影响的SGC-7901-siControl细胞。分别采用RT-PCR和Western blot技术检测非转染组、SGC-7901-siSnail、SGC-7901-siControl三组细胞Snail、α-平滑肌肌动蛋白(α-SMA)和E-cadherin表达,用Boyden chamber模型检测细胞侵袭能力。结果 SGC-7901-siSnail组与SGC-7901-nontransfection组相比,Snail和α-SMA表达显著减弱(P0.01),E-cadherin表达显著增强(P0.01),Boyden chamber穿膜细胞数显著减少(P0.01);SGC-7901-siControl组中Snail、α-SMA、E-cadherin表达、Boyden chamber穿膜细胞数分别和SGC-7901-nontransfection组比较无显著差异(P0.05)。结论通过RNA干扰阻滞Snail表达能有效地抑制SGC-7901细胞上皮-间充质转化及体外侵袭能力。Snail可能在胃腺癌上皮-间充质转化及侵袭过程中扮演重要角色,抑制Snail表达可能成胃腺癌治疗的可行策略。     

PDSW联合热疗可抑制胃癌NF-κB的表达

为探讨生理性深层海水(PDSW)联合热疗对人胃癌SGC-7901细胞体外和体内的抗癌作用,通过在体外培养人胃癌SGC-7901细胞株,随机分为实验组、对照组和空白组,实验组加PDSW,对照组加生理盐水,空白组不做其他处理,分别在37℃、40℃和43℃热疗温度下培养。同时,将SGC-7901细胞注射至裸鼠皮下,建立移植瘤模型,随机分为5组,至肿瘤长至直径为1 cm时,分别进行H_2O+RT、H_2O+40℃、H_2O+43℃、PDSW+40℃、PDSW+43℃处理,然后绘制裸鼠肿瘤生长曲线,观察裸鼠肿瘤组织切片,并用实时荧光定量PCR检测细胞和组织中NF-κB基因表达情况,Western blotting检测NF-κB蛋白的表达情况。研究发现,与其他处理相比,PDSW联合热疗处理后肿瘤生长缓慢,肿瘤细胞的增长明显受抑,细胞和裸鼠皮下移植瘤组织中NF-κB基因和蛋白的表达量较低。说明PDSW联合热疗能抑制胃癌SGC-7901细胞中NF-κB基因和蛋白的表达,从而有效抑制胃癌细胞的增殖。     

全反式维甲酸诱导胃腺癌细胞周期阻滞的作用机制

目的:研究全反式维甲酸(all trans retinoic acid,ATRA)对人胃腺癌细胞(SGC-7901)细胞周期阻滞的诱导作用并探讨其机制.方法:采用四甲基偶氮唑盐(MTT)方法检测ATRA对SGC-7901增殖的抑制;流式细胞术检测细胞周期,Western blot方法检测不同浓度ATRA处理后的SGC-7901细胞中Akt、P-Akt(Ser473)、P-Akt(Thr308)、p-GSK-3β(Ser9)和cyclin D1的表达情况.结果:10.9～10.5mol/L的ATRA作用SGC-7901细胞48 h,能显著抑制细胞增殖,其抑制率分别为10.2%±0.5%、15.3%±0.5%、17.0%±0.7%、28.4%±1.0%和36.9%±0.7%;G1期细胞比率随着ATRA浓度的增加而增加,呈明显的G1期阻滞;Western blot检测显示ATRA对细胞中Akt蛋白的表达没有明显影响,两种p-Akt蛋白的表达显著下调,ATRA显著降低细胞中cyclin D1和p-GSK-3β的表达.结论:ATRA可能通过抑制磷酸化Akt蛋白表达而减少p-GSK-3β的表达,从而减少cyclin D1的表达量,进而诱导SGC-7901细胞发生G1期阻滞.     

建立胃癌实验动物模型方法的研究

目的通过对人低分化胃癌细胞系SGC-7901状态和接种细胞数目的研究,建立良好的皮下接种胃癌的动物模型。方法采用腹腔注射SGC-7901细胞,使裸鼠形成腹水;光镜及电镜观察细胞状态;将购买的SGC-7901细胞以及形成腹水后的肿瘤细胞分别以1×108、1×107和1×106个进行裸鼠皮下接种,每组接种5只。观察其肿瘤形成时间、大小、状态及病理学变化。结果SGC-7901细胞接种裸鼠形成腹水后进行培养的肿瘤细胞增殖状态发生改变;购买的SGC-7901细胞以1×108及1×107接种裸鼠,在第21天肿瘤组织中央均出现出血和坏死;1×106的裸鼠第21天未见肉眼可见的肿瘤形成。腹水培养的肿瘤细胞,接种1×108的裸鼠在第21天肿瘤组织中央可见大面积的出血和坏死;接种1×107及1×106的裸鼠在第21天均未见出血和坏死,接种1×107的肿瘤组织体积较大。结论SGC-7901细胞接种裸鼠形成腹水后的细胞,更容易建立SGC-7901细胞皮下接种的胃癌动物模型,其中以接种细胞数为1×107的肿瘤生长较好,更适用于胃癌的实验研究。     

The translation factors of Drosophila melanogaster

Synthesis of polypeptides from mRNA (translation) is a fundamental cellular process that is coordinated and catalyzed by a set of canonical ‘translation factors’. Surprisingly, the translation factors of Drosophila melanogaster have not yet been systematically identified, leading to inconsistencies in their nomenclature and shortcomings in functional (Gene Ontology, GO) annotations. Here, we describe the complete set of translation factors in D. melanogaster, applying nomenclature already in widespread use in other species, and revising their functional annotation. The collection comprises 43 initiation factors, 12 elongation factors, 3 release factors and 6 recycling factors, totaling 64 of which 55 are cytoplasmic and 9 are mitochondrial. We also provide an overview of notable findings and particular insights derived from Drosophila about these factors. This catalog, together with the incorporation of the improved nomenclature and GO annotation into FlyBase, will greatly facilitate access to information about the functional roles of these important proteins.     

Model analysis of difference between EGF pathway and FGF pathway

The difference in time course of Ras and mitogen activated protein kinase (MAPK) cascade by different growth factors is considered to be the cause of different cellular responses. We have developed the computer simulation of Ras-MAPK signal transduction pathway containing newly identified negative feedback system, Sprouty, and adaptor molecules. Unexpectedly, negative feedback system did not profoundly affect time course of MAPK activation. We propose the key role of fibroblast growth factor receptor substrate 2 (FRS2) in NGF/FGF pathway for sustained MAPK activation. More Grb2-SOS complexes were recruited to the plasma membrane by binding to membrane-bound FRS2 in FGF pathway than in EGF pathway and caused sustained activation of ERK. The EGF pathway with high concentration of EGF receptor also induced sustained MAPK activation, which is consistent with the results in the PC12 cell overexpressing the EGF receptors. The simulated time courses of FRS2 knock-out cells were consistent with those of the reported experimental results.     

The association of human coagulation factors VIII,IXa and X with phospholipid vesicles involves both electrostatic and hydrophobic interactions

Blood coagulation factor X (FX) is converted to its active form (FXa) by a membrane bound multi-protein enzyme complex, comprised of factor VIII (FVIII), factor IXa (FIXa) and FX. Characterization of the molecular forces involved in the association of these proteins with phospholipids is crucial to understanding how these proteins bind to the lipid milieux of physiological membranes. In this report, the molecular forces involved in the association of FVIII, FIXa or FX with phospholipid vesicles (PLV) were characterized by ligand affinity chromatographic analyses. Treating FVIII-affinity columns with agents that disrupt electrostatic interactions caused elution of 15.2% of the total bound PLV, while agents that disrupt hydrophobic interactions caused elution of 84.8% of the total bound PLV. These results demonstrate that the association of PLV with FVIII is primarily hydrophobic. In contrast, the association of PLV with FIXa or FX is largely the result of electrostatic forces. This was established by observing that 71.3% and 78.9% of the total bound PLV was eluted from FIXa- and FX-affinity columns, respectively, by agents that disrupt electrostatic interactions. Of the total bound PLV, 28.7% and 21.2% were eluted from FIXa- and FX-affinity columns, respectively, by agents that disrupt hydrophobic interactions. These data demonstrate that hydrophobic forces play a heretofore unrecognized role in the association of PLV with FIXa or FX.     

凝血因子药物市场分析

凝血因子是一类特殊的药物,是血友病等血液疾病的治疗药物,目前已经成为血液制品的重要组成部分。国外已经有二十多种重组凝血因子药物上市,2015年全球重组凝血因子药物的市场规模已经达到78.54亿美元,未来还将持续增长,Baxalta公司的重组凝血因子产品销售额位居全球首位,达到28.40亿美元。国外有多种重组凝血因子处于研发阶段,其中长效重组凝血因子将成为新的市场增长点。国内各类凝血因子药物的批签发状况良好,且随着国家发展和改革委员会取消血液制品最高零售价,各类产品价格均有不同幅度增长,其中人纤原蛋白原增长幅度最高,达到189%。国产凝血因子市场空间巨大,但存在产品供给和研发力度不足等问题,发展受到限制,必须改革行业管理制度、提高血浆分离技术、加强重组产品研发。     

生长因子及其受体在淋巴瘤中异常表达的研究进展

生长因子是一类与受体结合后可以促进细胞增殖和调节细胞多项功能的多肽分子。生长因子及其受体信号通路包括Ras/MAPK、PI3K/AKT和STAT等不仅调控正常细胞的生物学行为,对恶性肿瘤细胞增殖、分化、转化和迁移也具有重要意义。研究发现多种生长因子如VEGF、PDGF和IGF及其受体在多种实体肿瘤如肺癌、乳腺癌、结肠癌中发现有异常表达,在淋巴瘤如DLBCL、PTCL、ML和NL中也存在异常的共同表达,提示在淋巴瘤中可能构成生长因子及其受体的自分泌/旁分泌环路。生长因子及其受体的表达对淋巴瘤患者的预后有一定指导意义,临床研究发现表达生长因子或其受体阳性患者比表达阴性患者有较差的临床预后。这可能与生长因子及其受体对淋巴瘤细胞的增殖、转移和耐药调控有关。目前生长因子及其受体已成为潜在的药物靶点,多种生长因子及其受体抑制剂在开发和临床试验中。本文就近年来生长因子及其受体在淋巴瘤中异常表达研究进展作简要综述。     

入侵生物福寿螺抗逆性的研究进展

福寿螺因具有较强的环境适应能力,在热带、亚热带地区大量繁殖,造成生态危害,被定义为恶性外来入侵物种。世界遭入侵的地区均采取了各种措施消灭福寿螺,但防治成效不大,因为其具有很强的抗逆性,如福寿螺能适应39℃的极端高温,能够忍受长达14d的干旱,抵抗pH4.5～9.4的酸碱侵蚀,并能抵抗化学药剂、细菌、寄生虫的威胁。因此,研究其抗逆性及其机制将有助于找到更有效的防治方法。本文综述了国内外有关福寿螺抗物理(低温、高温、干旱)、化学(酸碱性、药物)、生物(细菌和寄生虫、捕食者)因素的研究成果,旨在为防治福寿螺提供理论基础,也为进一步研究福寿螺的抗逆性提供参考。目前的研究表明,福寿螺的抗逆性由外界环境因子诱发,环境因子促成其生理状态,如代谢途径、糖类和氨基酸积累等的改变,这些变化由基因调控,可遗传给后代;外界环境因子还能引起福寿螺对逆境的行为适应性、调节产卵量等。     

我国促肝细胞生长因子的研究和应用

促肝细胞生长因子具有促进细胞增殖和生长的作用,国内外研究较多。我国对肝细胞生 长因子的研究和应用主要集中在肝源性促肝细胞生长因子上。在总结国内学者历年研究成果的 基础上,结合我国生产和应用的实际情况,对促肝细胞生长因子的来源、结构、理化性质、生产及 应用情况进行了综述,有助于更深入研究。     

Activation of blood coagulation factor VIIa with cleaved tissue factor extracellular domain and crystallization of the active complex

Exposure of blood to tissue factor leads to the formation of a high affinity tissue factor/factor VIIa complex which initiates blood coagulation. As a first step toward obtaining structural information of this enzyme system, a complex of active-site inhibited factor VIIa (F.VIIai) and soluble tissue factor (sTF) was prepared for crystallization. Crystals were obtained, but only after long incubation times. Analysis by SDS-PAGE and mass spectrometry indicated the presence of sTF fragments similar to those formed by proteolytic digestion with subtilisin (Konigsberg, W., Nemerson, Y., Fang, C., Lin, T.-C. Thromb. Haemost. 69:1171, 1993). To test the hypothesis that limited proteolysis of sTF facilitated the crystallization of the complex, sTF fragments were generated by subtilisin digestion and purified. Analysis by tandem mass spectrometry showed the presence of nonoverlapping N- and C-terminal sTF fragments encompassing more than 90% of the tissue factor extracellular domain. Enzymatic assays and binding studies demonstrated that an equimolar mixture of N- and C-terminal fragments bound to factor VIIa and fully restored cofactor activity. A complex of F.VIIai and sTF fragments was prepared for crystallization. Crystals were obtained using microseeding techniques. The best crystals had maximum dimensions of 0.12 × 0.12 × 0.6 mm and showed diffraction to a resolution of 3 Å. © 1995 Wiley-Liss, Inc.     

Acute Regulation of the Epidermal Growth Factor Receptor in Response to Nerve Growth Factor

PC12 cells possess specific receptors for both nerve growth factor and epidermal growth factor, and by an unknown mechanism, nerve growth factor is able to attenuate the propagation of a mitogenic response to epidermal growth factor. The differentiation response of PC12 cells to nerve growth factor, therefore, predominates over the proliferative response to epidermal growth factor. We have observed that the addition of nerve growth factor to PC12 cells rapidly produces a decrease in surface 125I-epidermal growth factor binding capacity. Unlike previously described nerve growth factor effects on 125I-epidermal growth factor binding capacity, which required several days of nerve growth factor exposure, the decreases we report occur within minutes of nerve growth factor addition: A 50% decrease in 125I-epidermal growth factor binding capacity is evident at 10 min. This rapid nerve growth factor response is concentration dependent; inhibition of 125I-epidermal growth factor binding is detectable at nerve growth factor levels as low as 0.2 ng/ml and is maximal at approximately 50 ng/ml, consistent with known ranges of biological activity. No demonstrable differences in the rate of epidermal growth factor receptor synthesis or degradation were observed in cells acutely exposed to nerve growth factor. Scatchard analysis revealed that acute nerve growth factor treatment decreased the number of both high- and low-affinity 125I-epidermal growth factor binding sites, while the receptor affinity remained unchanged. We have also investigated the involvement of various potential intracellular mediators of nerve growth factor action and of known intracellular modulatory systems of the epidermal growth factor receptor for their capacity to participate in this nerve growth factor activity.     

Fluorine and metals in Polygonum arenastrum Bor. from areas influenced by various types of industry

A herbaceous plant Polygonum arenastrum Bor. (=P. aequele Lindm., Polygonum aviculare ssp. aequale (Lindman) Ascherson & Graebner) (equal-leaved knotgrass), is a widespread, good coloniser, able to survive in wastelands where other species became extinct. Therefore, the bioindication abilities of this species for F, Cd, Co, Cr, Cu, Fe, Hg, Mn, Ni, Pb, and Zn were investigated. P.arenastrum was collected from 90 sites affected by six polluting factories: copper smelter, chlor-alkali plant, former ferrous-chromium smelter and active combustion smelter, power plant, cement plant and coke plant. Plant samples were collected at 0.75, 1.5, 3 and 4.5 km from each of these polluters in N, S, W and E directions. For comparison, a control site with 16 sampling points was selected in an area relatively free from pollution. Concentrations of Cr, Cu, Fe, Pb and Zn in shoots and roots positively correlated with those in soil (both total and plant-available). Cu, Pb and Zn in P.arenastrum were within the values reported in literature as toxic for other plants with the highest bioaccumulation factor (BF) from soil to shoots for Cr and the highest translocation factor (TF) from roots to shoots for Co and Pb for more and less polluted and control sites. Polygonum arenastrum has a potential to accumulate Cr even in sites with low chromium concentration in soil. Polygonum arenastrum does not show any form of shoot injury at increased levels of F and metals in its tissues. Based on these characteristics we conclude that the ubiquitous P.arenastrum may be utilised as a relevant indicator of contamination in industrial zones and may function as an early warning system of increased toxicity in the environment.