Stable silver nanoparticles–aptamer bioconjugates for cellular prion protein imaging

Combining the unique optical properties of metal nanoparticles and the specific recognition of aptamer,aptamer–nanoparticle conjugates have been extensively used in a wide range of applications,particularly multifunctional nanoparticles for cell detection and molecular imaging.Conventional conjugates prepared by chemisorption of monothiol-modified oligonucleotides onto nanoparticle surfaces suffer from a lack of stability when exposed to a variety of small molecules.If silver is used in place of gold,then this lack of stability is even more pronounced.In this study,we reported here the effective and facile strategy of preparing stable silver nanoparticle–aptamer conjugates by in situ generation of strong metal affinity capping ligands,dithiocarbamates modified anti-prion protein aptamer.The conjugates produced are stable and can withstand NaCl concentration at0.25 mol/L.Meanwhile,they could be applied in the cellular prion protein imaging successfully.     

基于核酸适配体Q5的石斑鱼虹彩病毒快速检测技术

石斑鱼虹彩病毒(Grouper iridovirus,SGIV)在不同鱼群之间迅速传播,其致死率极高,严重制约了我国海水石斑鱼(Epinephelus tauvina)养殖业健康可持续发展。为了及早发现和鉴定出病原,降低经济损失,促进渔业发展,开发方便快捷的石斑鱼虹彩病毒检测技术已迫在眉睫。本研究基于核酸适配体Q5,研发出一种核酸适配体Q5-荧光分子探针(Aptamer Q5-based fluorescent molecular probe,Q5-AFMP),并且对Q5-AFMP检测感染石斑鱼虹彩病毒的特异性,以及Q5-AFMP在细胞水平和组织水平检测石斑鱼虹彩病毒感染的灵敏度进行分析。研究结果表明:Q5-AFMP能够在细胞水平和组织水平高特异性检测出石斑鱼虹彩病毒的感染,而且具有较高的灵敏度。因此Q5-AFMP能够用于石斑鱼虹彩病毒的快速检测和诊断。     

基于适配体的胶体金色谱层析试纸条快速检测肌酸激酶同工酶

研究基于适配体的胶体金色谱层析试纸条快速检测肌酸激酶同工酶,用于揭示长期飞行下航天员潜在的心肌和肌肉损伤.利用SELEX方法筛选得到的肌酸激酶同工酶适配体,并优选出与肌酸激酶同工酶有较强亲和能力的核酸适配体应用于胶体金免疫层析试纸条,通过优化胶体金颗粒大小、缓冲液浓度等条件,成功建立了一种基于适配体的胶体金试纸法用于CK-MB的快速检测.实验结果表明,通过基于磁珠的核酸适配体筛选方法筛选出的与肌酸激酶同工酶有亲和力的核酸适配体,能够用于构建适配体-适配体夹心胶体金免疫层析试纸条并成功应用于82 nM以上的肌酸激酶同工酶的快速、可视化检测,同时,该免疫层析试纸条具有良好的特异性和稳定性,可用于未来空间医学即时检测.     

基于肺部动态增强MR影像的药物动力学定量分析

针对临床肺部对比剂动态增强磁共振成像(DCE-MRI)数据，提出一种新的药物动力学参数定量分析FCM-BRR方法.该方法基于参考区域模型(referenceregion，RR)，结合模糊C均值聚类(fuzzyC-mean，FCM)和B-样条(B-Spline)曲线拟合技术，解决了传统模型针对具有复杂血供系统的局限性，并且降低了噪声对结果的影响.利用FCM-BRR方法分析了5位病理证实的肺癌患者的临床DCE-MRI数据，检验了该方法的有效性和可行性.FCM-BRR方法给出的Ktrans参数图像反映了肿瘤组织和正常组织的异质性，并通过统计分析知正常组织和肿瘤组织之间具有显著差异(P<001).FCM-BRR方法能够直接利用常规临床数据定量分析病灶区域的药物动力学参数，在治疗评估肺部恶性肿瘤上具有参考意义.     

从福尔马林固定的癌组织中提取RNA的方法

目的:寻求从福尔马林固定的癌组织中提取RNA的合理方法.方法:运用Trizol法从福尔马林固定的胃癌组织中提取RNA,用分光光度计测定RNA的产量和纯度;通过RT-PCR对不同长度的管家基因-actin进行扩增.结果:100 bp和300 bp的-actin均能顺利扩增出特异性条带,500 bp的-actin未能扩增.结论:用Trizol法从福尔马林固定的癌组织中提取RNA是可行的;进行RT-PCR时应尽量设计小片段引物(100~300 bp)以获得更好的扩增效果.     

Intravenous delivery of a multi-mechanistic cancer-targeted oncolytic poxvirus in humans

The efficacy and safety of biological molecules in cancer therapy, such as peptides and small interfering RNAs (siRNAs), could be markedly increased if high concentrations could be achieved and amplified selectively in tumour tissues versus normal tissues after intravenous administration. This has not been achievable so far in humans. We hypothesized that a poxvirus, which evolved for blood-borne systemic spread in mammals, could be engineered for cancer-selective replication and used as a vehicle for the intravenous delivery and expression of transgenes in tumours. JX-594 is an oncolytic poxvirus engineered for replication, transgene expression and amplification in cancer cells harbouring activation of the epidermal growth factor receptor (EGFR)/Ras pathway, followed by cell lysis and anticancer immunity. Here we show in a clinical trial that JX-594 selectively infects, replicates and expresses transgene products in cancer tissue after intravenous infusion, in a dose-related fashion. Normal tissues were not affected clinically. This platform technology opens up the possibility of multifunctional products that selectively express high concentrations of several complementary therapeutic and imaging molecules in metastatic solid tumours in humans.     

Study on the specific interaction between angiogenin and aptamer by atomic force microscopy （AFM）

The specific interaction between angiogenin and aptamer has been investigated by using AFM. The specificity of the interaction is revealed by comparing the binding probability of aptamer to other elements in a series of control experiments. The results have shown that there is specific interaction force between angiogenin and aptamer. Moreover, the single molecular pull-off force between angiogenin and aptamer has also been determined using the Poisson statistical method to be 133.7±11.7 pN. These findings obtained are helpful to the better revelation of recognition mechanism between angiogenin and aptamer, which provided basis for further understanding the inhibition of the aptamer to angiogenic activity.     

一种从路由表推导自治系统关系的方法

提出一种利用边界网关协议(BGP)路由表推导自治系统(AS)关系的方法·通过分析AS关系对BGP输入和输出路由策略的约束,得到用AS关系序列表示的AS路径结构·根据AS关系序列上下文,利用AS路径构成规则推导AS关系·根据不同层次自治系统BGP路由表中AS路径的结构特点,对顶层AS和非顶层AS的BGP路由表分别使用不同的推导规则·推导中考虑了错误配置的影响,并且使用多种参数判断AS关系·利用公共可用BGP路由表测试算法,超过80%的AS关系可由顶层AS路由表直接导出·与同类算法结果对比验证了算法的有效性·     

Live birth after ovarian tissue transplant

Radiation and high-dose chemotherapy may render women with cancer prematurely sterile, a side-effect that would be avoided if ovarian tissue that had been removed before treatment could be made to function afterwards. Live offspring have been produced from transplanted ovarian tissue in mice and sheep but not in monkeys or humans, although sex steroid hormones are still secreted. Here we describe the successful transplantation of fresh ovarian tissue to a different site in a monkey, which has led to the birth of a healthy female after oocyte production, fertilization and transfer to a surrogate mother. The ectopically grafted tissue functions without surgical connection to major blood vessels and sets the stage for the transplantation of cryopreserved ovarian tissue in humans.     

Cellular inactivation by ultrasound

The lethal effect of ultrasound (US) on mammalian cells has received relatively little attention. Understandably, potential genetic aspects of US have been of prime concern to physicians who use US as a diagnostic tool; at the average power densities involved (<1 W cm(-2)) little, if any cell killing is to be expected. There have been sporadic attempts to use higher intensities ( approximately 1 W cm(-2)) as a treatment modality in cancer therapy, but those experiments seem to have been based on inadequate cellular studies. The effects of US usually were evaluated in terms of morphological criteria rather than on quantitative determination of the loss of viability as measured by colony formation. There are few reports of the effects of US on survival of mammalian cells, and none specifically examine hyperthermic interaction. With the increased interest in hyperthermia for tumour therapy, attention has been directed towards the use of ultrasound to achieve tumour heating. In preliminary experiments in which US was used to heat the EMT6 sarcoma and KHJJ carcinoma in mice, we found a high percentage of tumour cures with short (approximately 30 min) treatments at temperatures (43-44 degrees C) where in vitro results of hyperthermia-induced cell killing would not have led to a prediction of any cures. We therefore initiated an investigation of the effects of US on survival of Chinese hamster cells to see if direct cell killing by US could explain our in vivo results, or, as in the case of radiofrequency (RF) electromagnetic heating, we would be forced to invoke host response(8). In particular, we examined the thermal and non-thermal components of cellular inactivation by US. We report here that there is a definite non-thermal cytotoxic effect of US. Its relative contribution to cell killing is a highly nonlinear function of the temperature of the cellular milieu. The survival curves show clearly that, beyond an initial threshold, small changes in temperature and/or US intensity can give rise to impressive changes in survival values. The threshold nature of the data strongly suggests that by means of overlapping beams, ultrasound energy could be delivered to tumour tissue to achieve massive cell killings while sparing normal tissue outside the tumour volume to a degree far exceeding that of conventional techniques.     

常见肝癌细胞系的转铁蛋白受体表达差异分析及主动靶向载体效率比较

转铁蛋白受体1(transferrin receptor 1,TfR1)可介导细胞内吞过程,从而摄取与之特异结合的纳米颗粒,因此成为许多主动靶向型纳米载体的靶点。研究表明,肝癌细胞存在TfR1高表达现象,可作为肿瘤治疗纳米药物递送系统的关键性靶点。体外评价是TfR1靶向纳米载体的重要研究环节,然而肝癌细胞模型种类繁多,其TfR1表达水平可能存在一定差异。选择了几种常见的肝癌细胞系,包括HepG2、Hep3B、MHCC97-H以及Huh-1,分别从mRNA水平以及蛋白水平测定了细胞系TfR1的表达情况,考察了转铁蛋白(Tf)以及转铁蛋白核酸适配体(transferrin nucleic acid aptamer, Tf-APT)对不同细胞的亲和效率。同时,制备了包载紫杉醇的TfR1靶向脂质体,并考察其对不同细胞系的细胞生长抑制作用。结果表明,4种肝癌细胞系在mRNA水平以及蛋白水平均存在TfR1的表达差异;同时,体外抗肿瘤结果显示,不同肝癌细胞系对紫杉醇-TfR1靶向脂质体的敏感性也存在显著不同。     

Tissue repair and stem cell renewal in carcinogenesis

Cancer is increasingly being viewed as a stem cell disease, both in its propagation by a minority of cells with stem-cell-like properties and in its possible derivation from normal tissue stem cells. But stem cell activity is tightly controlled, raising the question of how normal regulation might be subverted in carcinogenesis. The long-known association between cancer and chronic tissue injury, and the more recently appreciated roles of Hedgehog and Wnt signalling pathways in tissue regeneration, stem cell renewal and cancer growth together suggest that carcinogenesis proceeds by misappropriating homeostatic mechanisms that govern tissue repair and stem cell self-renewal.     

体外培养肿瘤细胞生长的微量量热法研究

应用 2 2 77热活性检测仪测定了MKN 4 5细胞和SMMC 772 1细胞的热功率 时间曲线 ,通过观测化疗药物卡铂对曲线的影响 ,进一步研究了该药物对癌细胞增殖的抑制作用 .本实验所用的微量热测定的方法对于癌细胞生物学的研究有重要意义     

热固性树脂固化过程的在线测量和控制

为了研究生产过程中树脂固化行为的温度曲线,我们设计并且研制出一个完整的在线测量和控制系统KD1411。本文重点介绍了KD1411的以下三个主要特点:(1)增益可编程控制的微弱信号放大器的设计;(2)固态继电器的可编程控制;(3)在线不褪色显示。     

Magnetic resonance imaging of pH in vivo using hyperpolarized 13C-labelled bicarbonate

As alterations in tissue pH underlie many pathological processes, the capability to image tissue pH in the clinic could offer new ways of detecting disease and response to treatment. Dynamic nuclear polarization is an emerging technique for substantially increasing the sensitivity of magnetic resonance imaging experiments. Here we show that tissue pH can be imaged in vivo from the ratio of the signal intensities of hyperpolarized bicarbonate (H(13)CO(3)(-)) and (13)CO(2) following intravenous injection of hyperpolarized H(13)CO(3)(-). The technique was demonstrated in a mouse tumour model, which showed that the average tumour interstitial pH was significantly lower than the surrounding tissue. Given that bicarbonate is an endogenous molecule that can be infused in relatively high concentrations into patients, we propose that this technique could be used clinically to image pathological processes that are associated with alterations in tissue pH, such as cancer, ischaemia and inflammation.     

Synchrotron refractive-index microradiography of human liver cancer tissue

Three human liver tissue samples （-5 mm × 40 mm × 20 mm） were excised from a cancer patient＇s liver during surgery. The microradiology analysis was performed with a non-standard approach on a synchrotron. High-resolution refractive-index edge-enhanced microradiographs that cover a larger volume of the liver tissue sample were obtained. The cancer tissue and normal tissue could be clearly identified and distinguished based on their different textures. Furthermore, new blood vessel hyperplasia was found near the cancer area. Blood vessels with a diameter smaller than 20 μm could be identified. These findings were fully consistent with the histopathological examination or the same area. Microradiographs of the newly formed blood vessels at different angles were also obtained. This result shows that it is possible to further develop this approach into a technique of microradiographic imaging for clinic diagnosis of liver cancer at the early stage.     

The Syk tyrosine kinase suppresses malignant growth of human breast cancer cells

Syk is a protein tyrosine kinase that is widely expressed in haematopoietic cells. It is involved in coupling activated immunoreceptors to downstream signalling events that mediate diverse cellular responses including proliferation, differentiation and phagocytosis. Syk expression has been reported in cell lines of epithelial origin, but its function in these cells remains unknown. Here we show that Syk is commonly expressed in normal human breast tissue, benign breast lesions and low-tumorigenic breast cancer cell lines. Syk messenger RNA and protein, however, are low or undetectable in invasive breast carcinoma tissue and cell lines. Transfection of wild-type Syk into a Syk-negative breast cancer cell line markedly inhibited its tumour growth and metastasis formation in athymic mice. Conversely, overexpression of a kinase-deficient Syk in a Syk-positive breast cancer cell line significantly increased its tumour incidence and growth. Suppression of tumour growth by the reintroduction of Syk appeared to be the result of aberrant mitosis and cytokinesis. We propose that Syk is a potent modulator of epithelial cell growth and a potential tumour suppressor in human breast carcinomas.     

Delineation of prognostic biomarkers in prostate cancer

Prostate cancer is the most frequently diagnosed cancer in American men. Screening for prostate-specific antigen (PSA) has led to earlier detection of prostate cancer, but elevated serum PSA levels may be present in non-malignant conditions such as benign prostatic hyperlasia (BPH). Characterization of gene-expression profiles that molecularly distinguish prostatic neoplasms may identify genes involved in prostate carcinogenesis, elucidate clinical biomarkers, and lead to an improved classification of prostate cancer. Using microarrays of complementary DNA, we examined gene-expression profiles of more than 50 normal and neoplastic prostate specimens and three common prostate-cancer cell lines. Signature expression profiles of normal adjacent prostate (NAP), BPH, localized prostate cancer, and metastatic, hormone-refractory prostate cancer were determined. Here we establish many associations between genes and prostate cancer. We assessed two of these genes-hepsin, a transmembrane serine protease, and pim-1, a serine/threonine kinase-at the protein level using tissue microarrays consisting of over 700 clinically stratified prostate-cancer specimens. Expression of hepsin and pim-1 proteins was significantly correlated with measures of clinical outcome. Thus, the integration of cDNA microarray, high-density tissue microarray, and linked clinical and pathology data is a powerful approach to molecular profiling of human cancer.     

Recombinant sTRAIL induces cell death of tumor cell lines as well as primary cancer cells

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a new member of TNF family. It was reported that TRAIL could induce apoptosis of tumor cells but not normal cells in tissue culture system. To further study the biological activity and potential clinical significance, a recombinant soluble TRAIL (rsTRAIL) has been expressed stably in E. coli after transformation of pET28b vector containing the extracellular domain of TRAIL. The yield of rsTRAIL is approximately as high as 60% of whole bacterial proteins. The rsTRAIL, purified by Ni+ -agarose affinity chromatography, could remarkably trigger apoptosis at the concentrations of 0.1-1 μg/mL in all 7 tumor cell lines tested in vitro. However, this killing activity has not been observed in mouse fibroblast cell line (NIH3T3) as normal control. Further investigation shows that the rsTRAIL could also kill primary tumor cells isolated freshly from patients with cardiac cancer, breast cancer and malignant thymoma, while the normal human peripheral blood lymphocytes are not killed under the same conditions. These results provide new evidence that rsTRAIL could induce apoptosis of tumor cells specifically and it could be a new promising medicine for tumor therapy.