不同热休克蛋白在肾细胞癌组织中的表达及其意义

目的　研究HSPgp96、HSP90、HSP70在肾细胞癌组织中的表达及其相互关系。方法　应用免疫组化S P法检测 5 4例肾细胞癌组织标本中HSPgp96、HSP90、HSP70的表达情况。结果　在肾细胞癌组织中HSPgp96、HSP90、HSP70表达明显较正常肾组织增强 (P <0 0 5 ) ,阳性率分别为 88 9%、 85 2 %、 90 7% ,三者之间无明显差异性 (P >0 0 5 ) ,其表达程度均与肾细胞癌临床分期、病理分级及细胞类型无显著相关性 (P >0 0 5 )。结论　HSPgp96、HSP90、HSP70的高表达在肾细胞癌的发生、发展中起促进作用 ,尤其是在加强肿瘤抗原的呈递、表达上具有重要临床应用价值。     

热休克蛋白在高温致神经管畸形中的表达

目的：检测热休克蛋白在高温致神经管畸形中的表达状况,以探讨高温致神经管畸形的机制。方法：在高温致金黄地鼠神经管畸形的动物模型上,利用免疫组织化学(SABC法)方法,检测高温致神经管畸形中,热休克蛋白(HSP70和HSP90)在神经上皮细胞及周围间充质细胞中的表达状况;同时利用地高辛标记的寡核苷酸探针进行原位杂交,检测HSP70 mRNA和HSP90 mRNA在神经上皮细胞及周围间充质细胞中的转录状况。结果：高温处理后2h,鼠胚神经上皮细胞及周围间充质细胞HSP70、HSP90的表达与正常对照组相比明显增强,8h和16h的表达达到高峰,24h后与对照组水平一致。原位杂交结果显示,高温处理后2h神经上皮细胞及周围间充质细胞中出现HSP70 mRNA及HSP90mRNA杂交阳性信号,8h阳性信号最强,16h后阳性信号减弱,至24h后未见阳性信号。结论：高温可引起神经上皮细胞及其周围间充质细胞HSP70和HSP90应激性表达,这可能是胚胎受到高温作用后发生的一种保护性反应。     

汉滩病毒感染诱导热休克蛋白70表达

为了解汉滩病毒感染后细胞的应激反应及HSP70的表达与病毒复制的关系,在汉滩病毒A9株感染Vero-E6细胞后,用免疫组织化学及核酸分子原位杂交法,对细胞HSP70基因的表达进行了检测.结果表明,汉滩病毒感染细胞4h后即可诱导Vero-E6细胞表达HSP70,表达可持续至感染后5d,且HSP70在细胞内的分布也有改变.提示汉滩病毒可直接诱导HSP70的高表达.     

TNF-a在乳鼠心肌细胞中诱导HSP70蛋白表达的研究

热休克蛋白70 (HSP70) 在细胞修复、存活和维持细胞正常功能方面有着重要作用。作为分子伴侣,它起着心肌保护的作用。已经对重症心脏病人的心肌组织进行了蛋白组学研究,得到了HSP70在心衰病人心肌组织中较正常人心肌组织表达升高的结论,并且在血液中得到了进一步的验证。在进一步的离体细胞实验中用不同剂量的肿瘤坏死因子-alpha (TNF-α) 刺激乳鼠心肌细胞,以观察不同时间点HSP70的动态表达情况。培养乳鼠心肌细胞,分别对细胞进行热休克（42 ℃）、TNF-α和缺血缺氧处理,在不同的时间点收获细胞,以观察HSP70的动态表达情况。用免疫化学、ELISA以及Western blotting的方法对HSP70蛋白进行分析。结果表明,在正常对照细胞中基本没有阳性信号出现,而在经缺血缺氧、热休克（42 ℃）以及TNF-α处理的细胞中有明显的阳性表达。以上研究首次在乳鼠心肌细胞中证明TNF-α诱导的HSP70表达具有时间和浓度依赖性。通过运用TNF-α对HSP70蛋白表达影响的研究,初步推断HSP70的表达模式,为体内诱导产生HSP70从而发挥心肌保护作用的研究提供一定的理论基础。     

用量子点标记活细胞

量子点是一种具有纳米尺寸的半导体晶体。与传统的荧光染料相比,量子点拥有许多独特的光学特性,如宽的吸收谱、窄而对称的发射谱、耐漂白、亮度高和荧光寿命长等。由于其出色的光物理特性和相对较小的尺寸,量子点可作为生物学研究的荧光探针。随着量子点合成与修饰技术的发展,其在生物和医学领域的应用已从探索阶段逐步发展到了应用阶段。将量子点应用于活细胞标记,将为揭示细胞内的复杂生命现象提供全新的视野。该文重点介绍了量子点的荧光特性、用量子点标记活细胞所要克服的障碍及基本的标记策略和方法。     

固定剂、封片剂、温度及除菌方式对量子点标记细胞的影响

为了为利用量子点标记细胞、组织,进一步研究其功能提供新的方法,本实验观察了3种发射波长的量子点（quautum dost,QDs）对所标记的小鼠腹腔巨噬细胞和正常皮肤的影响。利用发射波长610mm的红色荧光水溶液（量子点610）、发射波长为523mm的绿色荧光水溶液（量子点523）和发射波长576nm的黄色荧光脂溶性溶液（量子点576）的3种量子点（5mg／ml）以及具有吞噬能力的小鼠腹腔巨噬细胞、正常皮肤为载体,观察不同的除菌方式、温度、封片剂及固定剂对量子点标记细胞、组织的影响,为量子点在生物体内的应用及在生物制片过程中对其性能的影响等研究奠定基础。     

HSP90 mRNA在胃癌和大肠癌中表达的研究

目的为探讨胃癌和大肠癌细胞HSP90 mRNA表达的特点.方法利用核酸原位杂交技术,对79例胃肠癌组织进行检测.结果表明HSP90 mRNA在胃癌和大肠癌中的阳性率分别为55.56%(15/27)和69.23%(36/52).mRNA表达与病理类型、分化程度和有否淋巴结转移有相关性.结论 HSP90 mRNA在胃肠癌中有较高表达,检测HSP90 mRNA可以作为提示预后的重要临床指标.     

量子点在生物学中的研究进展

量子点作为一种新型的荧光标记物近年来已在生物学中获得广泛应用。本文总结了量子点的主要光学特性,其中包括荧光激发和发射光谱特性、量子产额、光漂白特性和荧光寿命等。重点综述了量子点在细胞标记、活体和组织成像、组合标记和光动力学治疗等生物学中的应用及其最新研究进展。同时讨论了量子点在应用中可能存在的细胞毒性等主要问题,最后对量子点在生物学中的应用前景作了展望。     

褐飞虱HSP70与HSP90基因特性及温度诱导表达

[目的]研究不同温度条件下,褐飞虱HSP70和HSP70基因的表达变化,探索HSP蛋白在褐飞虱对温度胁迫适应中的作用。[方法]利用同源克隆方法获得HSP70A和HSP70基因序列,实时荧光定量PCR(qRT-PCR)检测HSPs基因在不同温度诱导下的表达量。[结果]褐飞虱HSP70A基因包含1 896 bp的ORF,编码631个氨基酸; HSP70基因包含2 193 bp的ORF,编码730个氨基酸。HSP70A在38℃高温下,表达量出现不同程度下降; HSP70B和HSP70在32℃及38℃高温下能够被诱导高表达。在低温诱导下,HSP70A及HSP70B的表达出现不同程度下降(HSP70A在15℃处理6 h及10℃处理2 h例外),HSP70表达量在低温下为显著或者极显著上升。[结论] HSP70B和HSP70在褐飞虱的高温胁迫下起重要的作用;在低温下则主要通过提高HSP70的表达来保护机体内细胞的正常生理代谢。     

Noninvasive imaging of quantum dots in mice

Quantum dots having four different surface coatings were tested for use in in vivo imaging. Localization was successfully monitored by fluorescence imaging of living animals, by necropsy, by frozen tissue sections for optical microscopy, and by electron microscopy, on scales ranging from centimeters to nanometers, using only quantum dots for detection. Circulating half-lives were found to be less than 12 min for amphiphilic poly(acrylic acid), short-chain (750 Da) methoxy-PEG or long-chain (3400 Da) carboxy-PEG quantum dots, but approximately 70 min for long-chain (5000 Da) methoxy-PEG quantum dots. Surface coatings also determined the in vivo localization of the quantum dots. Long-term experiments demonstrated that these quantum dots remain fluorescent after at least four months in vivo.     

Heat shock proteins and survivin: relationship and effects on proliferation index of retinoblastoma cells

Survivin and HSPs (heat shock proteins) are important anti-apoptotic proteins. However, limited research has been done regarding the collective effects of HSPs and survivin on the proliferative activities of RB cells. The purpose of this study was to narrow this gap by focusing on the expression of HSP70 and HSP90 and the interaction of these proteins with survivin. The proliferative activities of RB cells were analyzed by assessing the Ki-67 labeling index. Ki-67 recognizes a nuclear antigen expressed in all phases of the cell cycle except G(0) and early G(1), which makes it an excellent marker of cells in the proliferative phase. Immunohistochemical procedures were performed on retinal tissues from 43 RB patients who had undergone enucleation. Expression of HSP70, HSP90 and survivin was found in 65.12%, 86.05% and 62.79% of the cases respectively. No expression of any of these markers was found in normal retinal tissues. Expression of survivin was more frequent when HSP90 was detected than when HSP90 was not detected (P<0.05). The Ki-67 labeling index was higher in cases in which HSP90 or survivin was found than in cases in which neither protein was found (P<0.05). The Ki-67 labeling index was higher in cases positive for both HSP90 and survivin than in cases in which neither protein or only one protein was found (P<0.05). Expression of HSP70 neither correlated with that of survivin, nor had any significant effect on the Ki-67 labeling index (P>0.05). Although expression of HSPs and survivin and the Ki-67 labeling index did not correlate with histopathologic typing of RB (P>0.05), our findings demonstrate that expression of HSP90 correlates with that of survivin in RB and the co-existence of survivin and HSP90 probably plays an important role in cellular proliferation in RB. Further work is indicated to clarify the role of these processes in progression of RB.     

Turning all the lights on: quantum dots in cellular assays

Quantum dot materials are increasingly used in cellular assays, and offer a powerful and enabling complement to existing methods of labeling proteins, such as green fluorescent protein. These materials give researchers the ability to study specificity and functional responses in cellular systems, in a highly multiplexed manner, at either a molecular or cellular level. The recent literature bears witness to the increasing use of quantum dots for the investigation of chemicals on biological systems, and paves the way to the use of these assays for high-throughput analysis of functional responses in relevant models at scales including molecular, cellular and whole animal.     

体内光量子点可视化图像在脑肿瘤中的应用

恶性胶质瘤年发病率约为5/100,000。美国每年有超过14,000例的新发恶性脑胶质瘤患者。治疗主要以手术治疗为主,手术肿瘤的切除程度影响患者的预后。外科手术治疗脑肿瘤需要精确定位脑肿瘤组织在正常脑组织中的位置以便能够获得精确的组织活检和肿瘤的完全切除。量子点是稳定存在的,产生荧光的可视化半导体纳米晶体。静脉注射量子点伴随着网状内皮系统和巨噬细胞的隔离。巨噬细胞可渗入到肿瘤组织并且能够吞噬通过静脉注射的光量子来产生可视化的肿瘤标记。通过巨噬细胞介导,将光量子运输至肿瘤组织展现了一种新兴技术来标记术前肿瘤组织。由于肿瘤组织中的光量子可以被光学成像和光谱学工具来探测,因此在脑肿瘤组织活检和切除中可以为外科医生提供可视化得实时反馈。     

Molecular profiling of single cells and tissue specimens with quantum dots

Quantum dots are tiny light-emitting particles on the nanometer scale. They are emerging as a new class of biological label with properties and applications that are not available with traditional organic dyes and fluorescent proteins. Recent advances, as reported in Science and Nature Biotechnology, have led to quantum dot bioconjugates that are highly luminescent and stable. These bioconjugates raise new possibilities for studying genes, proteins and drug targets in single cells, tissue specimens and even in living animals.     

A self-assembled quantum dot probe for detecting beta-lactamase activity

This communication describes a quantum dot probe that can be activated by a reporter enzyme, beta-lactamase. Our design is based on the principle of fluorescence resonance energy transfer (FRET). A biotinylated beta-lactamase substrate was labeled with a carbocyanine dye, Cy5, and immobilized on the surface of quantum dots through the binding of biotin to streptavidin pre-coated on the quantum dots. In assembling this nanoprobe, we have found that both the distance between substrates and the quantum dot surface, and the density of substrates are important for its function. The fluorescence emission from quantum dots can be efficiently quenched (up to 95%) by Cy5 due to FRET. Our final quantum dot probe, assembled with QD605 and 1:1 mixture of biotin and a Cy5-labeled lactam, can be activated by 32microg/mL of beta-lactamase with 4-fold increase in the fluorescence emission.     

Preferential translation of heat shock mRNAs in HeLa cells deficient in protein synthesis initiation factors eIF-4E and eIF-4 gamma.

Expression of antisense RNA against eukaryotic translation initiation factor 4E (eIF-4E) in HeLa cells causes a reduction in the levels of both eIF-4E and eIF-4 gamma (p220) and a concomitant decrease in the rates of both cell growth and protein synthesis (De Benedetti, A., Joshi-Barve, S., Rinker-Schaffer, C., and Rhoads, R. E. (1991) Mol. Cell Biol. 11, 5435-5445). The synthesis of most proteins in the antisense RNA-expressing cells (AS cells) is decreased, but certain proteins continue to be synthesized. In the present study, we identified many of these as stress-inducible or heat shock proteins (HSPs). By mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by reactivity with monoclonal antibodies generated against human HSPs, four of these were shown to be HSP 90, HSP 70, HSP 65, and HSP 27. The steady-state levels of HSP 90, 70, and 27 were elevated in relation to total protein in AS cells. Pulse labeling and immunoprecipitation indicated that HSP 90 and HSP 70 were synthesized more rapidly in AS cells than in control cells. The accelerated synthesis of HSPs in the AS cells was not due, however, to increased mRNA levels; the levels of HSP 90 and 70 mRNAs either remained the same or decreased after induction of antisense RNA expression. Actin mRNA, a typical cellular mRNA, was found on high polysomes in control cells but shifted to smaller polysomes in AS cells, as expected from the general decrease in translational initiation caused by eIF-4E and eIF-4 gamma depletion. HSP 90 and 70 mRNAs showed the opposite behavior; they were associated with small polysomes in control cells but shifted to higher polysomes in AS cells. These results demonstrate that HSP mRNAs have little or no requirement in vivo for the cap-recognition machinery and suggest that these mRNAs may utilize an alternative, cap-independent mechanism of translational initiation.     

Heat shock protein 90 expression in Epstein-Barr virus-infected B cells promotes gammadelta T-cell proliferation in vitro

The aim of this study was to elucidate the in vitro response of gammadelta T cells to Epstein-Barr virus (EBV)-infected B cells and to determine whether EBV-induced heat shock proteins (HSPs) might serve as gammadelta T-cell stimulants. Cytofluorometric analysis revealed HSP90 cell surface expression in 12% of the EBV-immortalized B-cell population in all four of the B-cell lines tested. HSP27, HSP60, and HSP70 were not detected on the cell surface by cytofluorometry in these same B-cell lines. HSP90 and HSP60, but not HSP70 or HSP27, were detected on the cell surface after 125I cell surface labeling and immunoprecipitation with anti-human HSP monoclonal antibodies. In vitro kinetic studies indicated that gammadelta T cells increased at least twofold by day 11 postinfection in cultures of EBV-seronegative peripheral blood lymphocytes infected with EBV, whereas percentages of alphabeta T cells in these same cultures either decreased slightly or remained relatively unchanged in response to EBV infection. Addition of anti-human HSP90 monoclonal antibody to the EBV-infected lymphocyte cultures inhibited gammadelta T-cell expansion by 92%. The inhibition of gammadelta T-cell expansion by anti-HSP90 antibody was reversed upon treatment with exogenous HSP90. Taken together, these results indicate that HSP90 played an important role in the stimulation of gammadelta T cells during EBV infection of B cells in vitro and may serve as an important immunomodulator of gammadelta T cells during acute EBV infection.     

Quantum Dots in Cell Biology

Quantum dots are semiconductor nanocrystals that have broad excitation spectra, narrow emission spectra, tunable emission peaks, long fluorescence lifetimes, negligible photobleaching, and ability to be conjugated to proteins, making them excellent probes for bioimaging applications. Here the author reviews the advantages and disadvantages of using quantum dots in bioimaging applications, such as single-particle tracking and fluorescence resonance energy transfer, to study receptor-mediated transport.