用BIAcore系统研究西妥昔单克隆抗体与表皮生长因子受体的结合

目的：应用基于表面等离子体共振技术的BIAcore3000系统研究国产西妥昔单克隆抗体（cetuximab）C225与可溶性重组人表皮生长因子受体（EGFR）的结合能力,并与国外已上市的西妥昔单抗Erbitux相比较。方法：在CM5传感器芯片上设置2个通道,一个氨基偶联重组人EGFR作为检测通道,另一个不固定EGFR作为空白参比通道;以HBS溶液作为工作液,流速为10μL／min;活化与封闭芯片;再以10μL／min的流速分别以梯度浓度进样C225和Erbitux,每个浓度级别检测2次;获得结合动态图谱,拟合处理后用软件模块进行参数计算。结果：C225与可溶性重组人EGFR的结合动力学常数K^为4．00×10^8L／mol,KD为2．50×10^-9mol／L;而Erbitux与可溶性重组人EGFR的结合动力学常数KA为4．25×10^8L／mol,KD为2．35×10^-9mol／L。结论：在与可溶性重组人EGFR的结合能力上,C225与Erbitux有相似的结合动力学特性。     

Far-Field Raman Imaging of Short-Wavelength Particle Plasmons on Gold Nanorods

The surface plasmon fields of gold nanorods with a diameter of 100 nm and lengths of 1–5 m are imaged by using far-field Raman scattering of methylene blue adsorbed on the rods. When optically exciting the nanorods under total internal reflection with wave vector and electric field vector orientations along the rod axis, the plasmon field intensity along this axis is observed to be periodically modulated. This modulation is attributable to a beating of the exciting light wave and the nanorod plasmon mode. The plasmon wavelength deduced from the beat frequency is 379 nm, which is considerably smaller than the exciting laser wavelength of 647 nm. In general, Raman imaging is shown to be a powerful technique to probe local plasmon fields using far-field spectroscopy.     

Surface plasmon resonance imaging on a microchip for detection of DNA-modified gold nanoparticles deposited onto the surface in a non-cross-linking configuration

We report theoretical predictions and experimental observations of the reduced detection volume with the use of surface-plasmon-coupled emission (SPCE). The effective fluorescence volume (detection volume) in SPCE experiments depends on two near-field factors: the depth of evanescent wave excitation and a distance-dependent coupling of excited fluorophores to the surface plasmons. With direct excitation of the sample (reverse Kretschmann excitation) the detection volume is restricted only by the distance-dependent coupling of the excitation to the surface plasmons. However, with the excitation through the glass prism at surface plasmon resonance angle (Kretschmann configuration), the detection volume is a product of evanescent wave penetration depth and distance-dependent coupling. In addition, the detection volume is further reduced by a metal quenching of excited fluorophores at a close proximity (below 10nm). The height of the detected volume size is 40-70nm, depending on the orientation of the excited dipoles. We show that, by using the Kretschmann configuration in a microscope with a high-numerical-aperture objective (1.45) together with confocal detection, the detection volume can be reduced to 1-2attoL. The strong dependence of the coupling to the surface plasmons on the orientation of excited dipoles can be used to study the small conformational changes of macromolecules.     

Structure-function studies of mEGF: Probing the type Iβ-turn between residues 25 and 26

The interaction between epidermal growth factor (EGF) and its receptor molecule is not completely understood and has received much attention recently. Studies combining site-directed mutagenesis and NMR spectroscopy have identified a number of EGF residues that are required for activity and are believed to interact directly with the receptor. Instead of focusing on these residues, this study combines site-directed mutagenesis and NMR spectroscopy to probe the role of the type I-bend located between residues 25 and 26 of the N-terminal subdomain of the protein. Ser25 of murine EGF is replaced by Pro in an attempt to stabilize this turn conformation to produce a variant of mEGF with increased activity relative to that for the native protein. Ser25 is also replaced by Ala, which is found at position 25 in human EGF (hEGF), as a more conservative replacement. Receptor binding studies demonstrate that both mutations produce about a 30% reduction in binding affinity, which is shown to result from local changes within the loop or minor perturbations of residues neighboring the loop rather than from long-range perturbations of the-sheet of the N-terminal subdomain. The type I-turn appears to remain intact in both mutants; however, replacement with Pro seems to introduce more flexibility into this region of the protein. These results demonstrate that perturbation of this-turn has little effect on EGF-receptor interactions.Abbreviations EGF epidermal growth factor - h human - m murine - TGF type transforming growth factor - NMR nuclear magnetic resonance - [S2A]mEGF mEGF missing the N-terminal asparagine and with the serine at position 2 replaced by alanine - [S2A,S25A]mEGF and [S2A, S25P]mEGF replacement of serine at position 25 in [S2A]mEGF by alanine and proline, respectively - ¹²⁵I-mEGF ¹²⁵I-labeled mEGF - DMEM Dulbecco's modified Eagle's medium - FCS fetal calf serum - HEPES N-(2-hydroxyethyl)piperazine-N-2-ethanesulfonic acid - BSA bovine serum albumin - COSY correlated spectroscopy - DQCOSY double-quantum filtered COSY - NOESY nuclear Overhauser spectroscopy - NOE nuclear Overhauser effect - TOCSY total correlation spectroscopy - ³ J(H-H^N) vicinal spin-spin coupling constant between amide proton and -proton - DSS 2,2-dimethyl-2-silapentane-5-sulfonate - chemical shift in ppm - ppm parts per million,     

Characterization of multilayer-enhanced surface-enhanced raman scattering (SERS) substrates and their potential for SERS nanoimaging

Multilayer gold surface-enhanced Raman scattering (SERS) substrates, which consist of continuous gold films that are separated by self-assembled monolayers (SAMs) and cast over 430-nm diameter silica nanospheres on a glass slide, have been evaluated as a means of further enhancing the SERS signals produced from conventional metal film over nanostructure substrates. Evaluation of the effect of various SAMs, with different terminal functional groups, on the SERS enhancement factor were measured and compared to conventional single-layer gold film over nanostructure substrates, revealing relative enhancements as great as 22.4-fold in the case of 2-mercapto-ethanol spacer layers. In addition to evaluation of the effect of different terminal functionalities, the effect of spacer length was also investigated, revealing that the shorter chain length alcohols provided the greatest signals. Employing the optimal SERS multilayer geometry, SERS nanoimaging probes were fabricated and the SERS enhancement factor and variability in enhancement factor were measured over the SERS active imaging area, providing absolute enhancements similar to previous silver-based SERS nanoimaging probes (i.e., 1.2 × 10⁸). Varying the size of the multilayer gold islands that were deposited on the tip of the SERS active nanoimaging probe, it is possible to tune the optimal SERS excitation wavelength accurately and predictably over the range of approximately 450 to 600 nm, without coating the entire surface of the probe and significantly reducing the transmission and resulting signal-to-noise ratio of the images obtained.     

Colloidal Synthesis of Plasmonic Metallic Nanoparticles

Solutions of Ag and Au nanoparticles are strongly colored because of localized surface plasmon resonance in the UV/visible spectral region. The optical properties of these nanoparticles may be tuned to suit the needs of the application. This article summarizes our work in recent years on the solution synthesis of nanoparticles with tunable optical properties. The systems of interest include zero-dimensional bimetallic Ag–Au nanoparticles with different structures, one-, two-, and three-dimensional anisotropic monometallic Ag or Au nanoparticles. All of these nanosystems were prepared from colloidal synthesis through simple changes in the synthesis conditions. This is a demonstration of the versatility of colloidal synthesis as a convenient scalable technique for tuning the properties of metallic nanoparticles. Zhang, Tan, and Xie contributed equally to this article     

结肠癌细胞株和组织中EGFR、p-EGFR和p-IGFR-1β的表达及意义

目的：研究EGFR、p-EGFR和p-IGFR-1β在结肠癌细胞株的表达及其与吉非替尼敏感性的关系,以及在肿瘤组织中的表达状况、相关性及其与临床病理参数的关系。方法：以western blot检测EGFR、p-EGFR和p-IGFR-1β在结肠癌细胞株和组织中的表达,以Quantity One软件测量各蛋白条带的灰度值。结果：EGFR、p-EGFR在Lovo细胞中表达量最高（P〈0.05）,EGFR在SW620细胞中的表达最低（P〈0.05）,在HCT116、HT29、LS174T和SW480细胞中的表达差异不显著。p-IGFR-1β在Lovo、HT29和SW480细胞中表达较弱,且在Lovo细胞中表达更弱,但在HCT116、LS174T和SW620细胞中的表达均较强（P〈0.05）。结肠癌组织中EGFR表达率为55.6%（10/18）,癌旁正常组织为37.5%（3/18）,肿瘤组织中有表达增高的趋势;p-EGFR在结肠癌组织表达率为33.3%（6/18）,较正常组织的5.6%（1/18）明显增高（P〈0.05）;p-IGFR-1β在结肠癌组织表达率为44.4%（8/18）,较正常组织的11．1％（2／18）明显增高（P〈0．05）。EGFR、P-EGFR和P-IGFR-1β相互之间的表达无明显相关性;EGFR、P-EGFR表达与各临床病理参数无明显相关;P．IGFR-1β在淋巴结转移患者中的表达显著增高（P〈0．05）,且在分期晚的患者中也可观察到这种趋势。结论：结合我们以往的研究结果,P．EGFR表达高伴p-IGFR-1β表达低提示结肠癌细胞对吉非替尼的敏感性好。P-EGFR和P—IGFR-1β在结肠癌组织中的表达较正常组织明显增高,且p-IGFR．1B表达高可能预示结肠癌患者有较高的转移风险。     

Different Epidermal Growth Factor Receptor (EGFR) Agonists Produce Unique Signatures for the Recruitment of Downstream Signaling Proteins

The EGF receptor can bind seven different agonist ligands. Although each agonist appears to stimulate the same suite of downstream signaling proteins, different agonists are capable of inducing distinct responses in the same cell. To determine the basis for these differences, we used luciferase fragment complementation imaging to monitor the recruitment of Cbl, CrkL, Gab1, Grb2, PI3K, p52 Shc, p66 Shc, and Shp2 to the EGF receptor when stimulated by the seven EGF receptor ligands. Recruitment of all eight proteins was rapid, dose-dependent, and inhibited by erlotinib and lapatinib, although to differing extents. Comparison of the time course of recruitment of the eight proteins in response to a fixed concentration of each growth factor revealed differences among the growth factors that could contribute to their differing biological effects. Principal component analysis of the resulting data set confirmed that the recruitment of these proteins differed between agonists and also between different doses of the same agonist. Ensemble clustering of the overall response to the different growth factors suggests that these EGF receptor ligands fall into two major groups as follows: (i) EGF, amphiregulin, and EPR; and (ii) betacellulin, TGFα, and epigen. Heparin-binding EGF is distantly related to both clusters. Our data identify differences in network utilization by different EGF receptor agonists and highlight the need to characterize network interactions under conditions other than high dose EGF.     

表面增强拉曼散射技术无标记检测乳腺癌细胞来源外泌体

外泌体含有释放细胞的特异性物质,反映了释放细胞的组成成分,成为液体活检的重要物质。为了鉴别正常乳腺上皮细胞和乳腺癌细胞来源的外泌体,本研究采用表面增强拉曼(SERS)技术检测乳腺癌细胞MCF-7和人正常乳腺上皮细胞MCF-10A来源的外泌体,并且对比两种细胞外泌体的SERS光谱,筛选出相应的特征拉曼光谱。结果发现在800~1800 cm-1区域内,相对MCF-10A细胞,MCF-7来源的外泌体中归属于核酸的拉曼峰相对强度明显增高,且部分脂类峰强有所降低或部分特征峰消失,同时发现MCF-7还表现出其自己独特的拉曼谱型。此结果表明SERS技术可高灵敏度检测不同细胞来源的外泌体细微分子变化,可区分肿瘤细胞来源的外泌体与正常细胞来源的外泌体。SERS技术可作为癌症的早期检测和诊断的一种快速、无标记和无损的方法。     

Localized surface plasmon resonance based gold nanobiosensor: Determination of thyroid stimulating hormone

An immunoassay method based on the peak shift of the localized surface plasmon resonance (LSPR) absorption maxima has been developed for the determination of the thyroid stimulating hormone (TSH) in human blood serum. The anti-TSH antibody was adsorbed on the synthesized gold nanoparticles by electrostatic forces. The efficiency of the nanobiosensor was improved by optimizing the factors affecting the probe construction such as the pH and the antibody to gold nanoparticles ratio. Dynamic light scattering was applied for the characterization of the constructed probe. The amount of peak shift of the LSPR absorption maxima was selected as the basis for determination of TSH antigen. The linear dynamic range of 0.4–12.5 mIU L⁻¹ and the calibration sensitivity of 1.71 L mIU⁻¹ were obtained. The human control serum sample was analyzed for TSH by constructed nanobiosensor and the acceptable results were obtained.     

EGFR和KDR在胃癌组织中的表达及临床意义

探讨表皮生长因子受体（epidermal growth factor receptor,EGFR）和含激酶插入区受体（kinase-insert domain-containing receptor,KDR）的表达与胃癌临床病理特征的关系及其临床意义.应用免疫组织化学PV法,检测60例胃癌组织中EGFR和KDR的表达情况,并且分析其与临床病理特征的关系.胃癌组织中EGFR和KDR阳性表达率分别为48.3%（29/60）、63.3%（38/60）,二者的阳性表达与浸润深度、淋巴结转移、TNM分期相关（P〈0.05）,并且存在共表达现象,表达强度呈正相关（rs=0.664,P〈0.05）.EGFR和KDR在胃癌的进展中起协同作用,为靶向EGFR和KDR生物治疗制剂在胃癌生物治疗中的应用及联合应用提供了一定的理论依据.     

Epidermal growth factor induces changes of interaction between epidermal growth factor receptor and actin in intact cells

The epidermal growth factor receptor (EGFR) is a cyto-skeleton-binding protein. Although purified EGFR can interact with acting in vitro and normally at least 10% of EGFR exist in the insoluble cytoskeleton fraction of A431 cells, interaction of cytosolic EGFR with actin can only be visualized by fluorescence resonance energy transfer when epidermal growth factor presents in the cell medium. Results indicate that the correct orientation between EGFR and actin is important in the signal transduction process.     

HER2、VEGF和MVD与胃癌患者临床病理特征的关系

目的：探讨HER2、VEGF的表达和MVD水平及这三者与胃癌的临床病理和生物学特性的关系。方法：胃癌患者26例,其中14例患者接受了全胃切除术,12例患者行胃癌活检,在癌组织中心获得有效标本设为胃癌组,以距离癌组织中心5cm的正常癌组织为正常组。RT-PCR法检测两组胃组织中HER2mRNA的表达;免疫组织化学检测胃组织中VEGF的表达;计算各组微血管密度。结果：与正常组相比,胃癌组HER2mRNA表达明显增加;正常组胃组织VEGF平均灰度值为21．51±4．64,而胃癌组胃组织VEGF平均灰度值为167．23±18．85,两组相比有明显差异;正常组胃组织MVD为13．44±5．34个,与正常组相比,胃癌组胃组织MVD水平（65．74±9．87个）明显增高;HER2与患者TNM分期成正相关,VEGF与患者性别及TNM分期均成正相关,而MVD与患者临床病理特征无相关性。结论：血管内皮生长因子的表达及与胃癌患者临床病理特征的相关性表明,HER2、VEGF两个分子生物标志物在肿瘤的发生发展中具有重要作用,其能够被用来作为预测的侵袭性胃癌预后的重要参数。