乳腺癌分子分型用荧光分子影像探针

目前,乳腺癌的临床诊断就是结合病理类型和分子分型 (免疫组化)。该方法是有创的,而且不能原位、实时地展现关键生物分子与临床关键信息之间的关系。本文介绍了利用分子影像技术对乳腺癌进行分子分型检测的最新研究进展。荧光成像灵敏度高,且不过分依赖图像分析,只需相应的荧光分子探针,即可实时、定量或半定量、多通道地得到肿瘤组织分子分型信息。因此,研制安全高效、组织穿透力强的近红外荧光分子探针是未来荧光成像技术和乳腺癌分子分型研究的重点。     

荧光分子成像的探针技术及其应用

近几年,随着生物和基因技术的发展,荧光探针的获取手段越来越丰富.荧光探针的发展使得荧光分子成像技术及其应用备受关注.借助于荧光探针,可以对目标分子、蛋白质、基因等特异性成像,从而实现分子、蛋白、基因等的精确定位与分析,进一步实现疾病的早期诊断与治疗.主要对荧光分子成像技术中用到的荧光探针技术以及在生物医学领域的应用进行概述.     

力传导中亚细胞水平的分子FRET成像

体内细胞受到含有化学和力学因素的生理和病理生理的刺激,故研究这些因素在细胞和器官水平如何调节功能就尤为重要。有关细胞和器官对化学因素的反应已开展诸多研究,而力学因素的影响却鲜有报道。近年来,荧光蛋白和显微镜技术的发展已成为阐明力传导过程的有用工具,先进的信号活细胞成像技术促进了力学生物学中分子机制的时空因素研究。本文综述荧光蛋白的基本知识以及其在生物学研究中的应用,特别讨论了以荧光共振能量迁移(fluorescence proteins and microscopy,FRET)技术为基础的生物传感器的发展和特征。基因编码的FRET生物传感器能够实现分子时空活动的成像和定量,使得活细胞中生物化学信号在力学刺激下的反应和传导可视化。同时,本文重点阐述分子水平力学刺激下的活细胞信号传导。     

一种水溶性稀土上转换荧光纳米探针及其活体荧光成像研究

目的制备一种水溶性稀土上转换荧光纳米探针NaYF4∶Yb3＋/Er3＋,用于动物活体荧光成像。方法采用三氟乙酸盐热分解法合成油溶性稀土上转换荧光纳米探针NaYF4∶Yb3＋/Er3＋,然后选用柠檬酸或L-半胱氨酸作为修饰剂,置于油-水两相中加热到100℃进行配体交换,离心纯化得到水溶性产物。并对所制备的上转换荧光纳米微粒进行X射线衍射测试（XRD）、X射线光电子能谱分析（XPS）、透射电子显微镜及红外和荧光光谱等表征,确定稀土上转换荧光纳米探针的组成、形态及修饰后该纳米探针的性质。最后将修饰后的水溶性稀土上转换荧光纳米探针作为探针应用于小鼠的活体成像。结果制备的稀土上转换荧光纳米探针NaYF4∶Yb3＋/Er3＋为立方相、平均粒径约21.3 nm的多边形结构,分别在540 nm和660 nm处发出荧光。用柠檬酸或L-半胱氨酸修饰后的稀土上转换荧光纳米探针可以稳定地分散在水中;并且修饰后的稀土上转换荧光纳米探针的粒径、晶相及荧光性质没有发生改变;红外光谱表明,该纳米探针的表面成功引入了修饰剂;该荧光纳米探针用于小鼠活体成像荧光信号清晰可见。结论研制出了一种用柠檬酸或L-半胱氨酸表面修饰的、水溶性稀土上转换荧光纳米探针NaYF4∶Yb3＋/Er3＋,表现出了较好的动物活体成像应用前景。     

肺癌靶向多肽荧光分子探针的研制及其应用

目的:选择能与整合素α3受体结合的小分子多肽cNGQGEQc-L作为靶向载体，将羧基荧光素（FAM）与cNGQGEQc-L连接构建荧光分子探针，通过荧光成像探讨荧光多肽分子探针用于肺腺癌显像的可行性。方法:利用倒置荧光显微镜观察FAM-cNGQGEQc-L与肺腺癌A549细胞结合部位，流式细胞仪检测荧光多肽与A549细胞的竞争抑制实验，观察FAM-cNGQGEQc-L随浓度的增加与肺腺癌A549细胞结合能力的变化情况。通过小动物活体成像仪，观察荧光多肽在荷瘤裸鼠体内的生物分布特点。结果:倒置荧光显微镜显示荧光多肽cNGQGEQc-L能与A549细胞结合，结合部位在细胞膜和细胞质中。流式细胞仪测试结果证明荧光多肽与A549细胞的结合具有特异性和饱和性，当FAM-cNGQGEQc-L浓度为0.125 mmol/L时，荧光多肽与A549细胞的结合趋近饱和。荷瘤裸鼠活体成像显示移植瘤能够摄取荧光多肽，且荧光多肽通过泌尿系统和胆道系统排泄。结论:体外、体内荧光实验结果显示，荧光多肽分子探针FAM-cNGQGEQc-L可与肺腺癌A549细胞、肺癌移植瘤结合，能够特异性靶向肺腺癌。     

活细胞内单分子视踪检测研究进展

对细胞生物学与分子生物学中有关分子事件和相互作用的认识,大部分都是在非活体条件下、忽略分子事件全过程所得到的研究结果,并且这些研究结果是基于被研究的所有单分子在相同的环境以相同的方式运动这个不真实的假设.随着量子点(QDs)等新的荧光物质和抗体模拟物等小分子抗体连接后制备成的新型探针的出现,以及转导外源性荧光探针进入活细胞内的各种方法的发展,再加上优良的荧光成像系统和快速灵敏的荧光采集系统在生命科学中的应用,使得视踪活细胞单个生物分子的轨迹、运动及多种单分子的相互作用成为现实.单分子水平的视踪研究为细胞生物学和分子生物学的研究打开了新的篇章.概述了目前活细胞内单分子视踪技术的研究进展并评价了其发展前景.     

用于荧光分子断层成像的小动物躯干部分三维表面轮廓重建研究

目的:基于CCD的荧光分子断层成像(FMT)近年来取得了快速发展,非接触式荧光分子断层成像技术(FMT)需要获取成像对象的三维表面轮廓,用于荧光团浓度重建的前向模型.方法:提出一种在白光条件下基于反投影法的三维轮廓提取方法,在获取360°的CCD照片后,滤波反投影重建断层,经过相应的图像平滑处理,得到轮廓边界,校准后重建出表面轮廓.结果:仿体实验和小动物实验结果表明,该方法具有毫米量级的重建精度.结论:该方法能够很好地重建躯干部分表面三维轮廓,较其他表面轮廓提取方法简单、实用,而且能够顺利地结合到荧光成像的工作流程中去,不需要单独成像,保证了整个成像过程的连续性.     

基于石墨烯场效应晶体管的新型冠状病毒抗原检测技术

目的建立一种简易、灵敏且快速的新型冠状病毒(2019 novel coronavirus, 2019-nCoV)抗原检测方法。方法构建石墨烯场效应晶体管器件, 并对传感器沟道材料进行探针分子修饰。在不同浓度2019-nCoV刺突蛋白溶液中进行电学信号的测试, 从而将抗原抗体特异性识别的生物信号转化为电学信号。结果抗体探针通过连接分子成功固定在石墨烯表面, 特异性靶向目标待测物, 从而确保传感器件的检测准确性。测试不同目标物浓度条件下石墨烯场效应晶体管的转移曲线得到相应的狄拉克点偏移, 最低检测浓度达到3.6×10-17 g/ml, 器件响应时间低至3 min。结论本研究初步建立了利用石墨烯场效应晶体管进行灵敏、高效且简便的2019-nCoV抗原检测方法。     

利用荧光探针Fluo-4检测胸腺细胞内钙离子浓度变化

利用荧光显微数码成像系统测量荧光探针Fluo-4荧光强度的改变,建立动态检测细胞内钙离子浓度的方法.用荧光探针Fluo-4/AM标记原代培养小鼠胸腺细胞,以KCL刺激胸腺细胞去极化,并打开细胞膜上电压依赖性钙通道,细胞内荧光强度会发生改变.利用荧光显微数码成像系统动态监测Fluo-4荧光强度的改变可分析计算钙离子浓度.本方法灵敏度高,能实时监测细胞内钙离子浓度的变化.     

力传导中亚细胞水平的分子FRET成像（英文）

体内细胞受到含有化学和力学因素的生理和病理生理的刺激,故研究这些因素在细胞和器官水平如何调节功能就尤为重要。有关细胞和器官对化学因素的反应已开展诸多研究,而力学因素的影响却鲜有报道。近年来,荧光蛋白和显微镜技术的发展已成为阐明力传导过程的有用工具,先进的信号活细胞成像技术促进了力学生物学中分子机制的时空因素研究。本文综述荧光蛋白的基本知识以及其在生物学研究中的应用,特别讨论了以荧光共振能量迁移(fluorescence proteins and microscopy,FRET)技术为基础的生物传感器的发展和特征。基因编码的FRET生物传感器能够实现分子时空活动的成像和定量,使得活细胞中生物化学信号在力学刺激下的反应和传导可视化。同时,本文重点阐述分子水平力学刺激下的活细胞信号传导。     

Recent advances in fluorescent labeling techniques for fluorescence microscopy

Tremendous progress in recent computer-controlled systems for fluorescence and laser-confocal microscopy has provided us with powerful tools to visualize and analyze molecular events in the cells. Various fluorescent staining and labeling techniques have also been developed to be used with these powerful instruments. Fluorescent proteins such as green fluorescent protein (GFP) allow us to directly label particular proteins of interest in living cells. This technique has been extended over a large area of cell biology, and a variety of fluorescent protein-derived techniques have been developed to visualize the functions and conditions of the molecules within living cells. In this review, we summarize the techniques for fluorescent staining and labeling for recent fluorescence microscopy.     

基于Monte Carlo荧光分子断层成像前向问题的研究

随着生物和光学技术的成熟，近红外荧光成像得到了飞速发展。荧光分子断层成像是以特定波长的光去激发荧光染料，使其吸收入射光，并发出波长长于入射光的近红外荧光。前向问题是进行光学成像研究的关键环节，可以指导实验以及重建算法。本文提出了一种基于Monte Carlo方法的荧光分子断层成像的光子传输模型，重点研究了激发光激发荧光的过程以及光子在生物体内的传播过程，在已知激发光源、传播介质、荧光物质等参数的条件下，得到荧光光子在CCD探测器上的分布情况，并且分别用理论和实验两种方法对算法的仿真结果进行了验证。     

Engineered multifunctional nanomaterials for multimodal imaging of retinoblastoma cells in vitro

Multifunctional nanoparticles are next generation materials that can be simultaneously used for imaging, diagnosis, and delivery of drugs. However, materials intended for cancer diagnosis need to be investigated for its cell uptake, toxicity, and effectiveness. In the current work, we have synthesized fluorescent iron oxide nanoparticles and evaluated its efficacy against retinoblastoma cell imaging. The iron oxide nanoparticles were synthesized and stabilized using oleic acid. Sulforhodamine B was adsorbed onto albumin over the oleic acid-capped iron oxide nanoparticles. Our results demonstrated good cell uptake in a time-dependent manner and nanoparticles were found to localize in the cytosol. Further, the nanoparticles exhibited excellent negative contrast in magnetic resonance imaging (MRI) experiments and with no cytoxicity (5–100?μg/mL iron oxide nanoparticles) to both normal as well as cancer cells demonstrating its biocompatibility. Thus, this novel material integrates the ability to image tissues with high sensitivity by MRI and specifically visualize Y79 retinoblastoma cells by fluorescence imaging with no toxicity.     

硫氧还蛋白和氧化/还原因子的融合荧光蛋白载体的构建及在293T细胞中的分布

目的构建硫氧还蛋白和氧化/还原因子的融合荧光蛋白真核表达载体,并在293T细胞中得到表达和定位。方法以RT-PCR方法从PC12细胞中克隆氧化/还原因子(APE/ref-1)的cDNA,然后亚克隆构建APE/ref-1-GFP融合真核表达载体。以PCR方法将质粒pQE30-TRX上的硫氧还蛋白cDNA亚克隆到pDsred1-1质粒上,然后将硫氧还蛋白-DsRed融合基因序列亚克隆到pCMV5质粒上,构建硫氧还蛋白-DsRed融合真核表达载体.通过磷酸钙转染293T细胞,以荧光显微镜分析融合蛋白的表达及其亚细胞定位。结果成功构建了硫氧还蛋白-DsRed和APE/ref-1-GFP融合表达载体,并在293T细胞中得到表达,APE/ref-1-GFP融合蛋白定位在293T细胞核内;硫氧还蛋白-DsRed融合蛋白定位在293T细胞质和细胞核内。结论为进一步研究硫氧还蛋白和APE/ref-1之间的动态相互作用奠定基础。     

Effect of pre- and postganglionic nerve divisions on normal postnatal and hydrocortisone-induced development of small intensely fluorescent cells in rat superior cervical ganglion

The left superior cervical ganglion of 3- or 23-day-old rats was subjected to pre- and/or postganglionic nerve division or sham operation, while the right ganglion was left intact. The animals were killed 20 or 60 days after the operation. Some animals were injected with 20 mg/kg hydrocortisone daily for 7 days and killed on the 8th day.Fluorescence microscopical examination revealed a normal postnatal increase in the number of small intensely fluorescent cells/ganglion after pre- or postganglionic nerve division, in spite of marked decreases in the volume of the operated ganglia. Combined pre- and postganglionic nerve division, which caused a dramatic loss of ganglion volume, entirely prevented the postnatal increase in the number of small intensely fluorescent cells. Hydrocortisone caused a large increase in the number of small intensely fluorescent cells both in intact and operated ganglia, including those in whom both pre- and postganglionic nerves had been divided.It is concluded that combined pre- and postganglionic denervation, in contrast to either operation alone, prevents the normal proliferation of the small intensely fluorescent cells possibly by causing an extensive loss of principal nerve cells which deprives the small intensely fluorescent cells of their normal contacts with the principal cells. Since the increase in the number of small intensely fluorescent cells due to hydrocortisone injections was not prevented by pre- and postganglionic denervation it must be due to a mechanism different from that responsible for the formation of small intensely fluorescent cells during normal postnatal development.     

Increase in the number of non-neuronal cells in superior cervical ganglia of developing rats after contralateral ganglionectomy

The left superior cervical ganglion of 3-day-old rats was subjected to preganglionic nerve division, ganglionectomy, or sham operation, while the right ganglion was left intact. Thirty days later, both the left and the right ganglia were perfusion-fixed and examined for weight and volume, as well as for the number and the density of the principal nerve cells and the non-neuronal cells. The small intensely fluorescent cells were counted from a separate set of freeze-dried ganglia.Unilateral preganglionic nerve division caused in the left operated side a significant loss of ganglion weight and volume due to a decreased number of non-neuronal cells, while no significant changes occurred in the right intact ganglion. Unilateral left ganglionectomy caused a significant increase in the mean ganglion weight and in the number and the density of the non-neuronal cells in the right intact ganglion, while the number and the density of the principal nerve cells and the small intensely fluorescent cells were not affected by this operation.It is suggested that normal development of the ganglionic satellite cells requires the presence of normally innervated principal cells. Furthermore, unilateral ganglionectomy induces a greater than normal proliferation of the satellite cells contralaterally, possibly by causing an increase in the activity of the contralateral ganglion.     

GFP和RFP报道基因在人体不同细胞中转导率的比较研究

目的 比较慢病毒载体介导的绿色荧光蛋白（GFP）和红色荧光蛋白（RFP）报道基因在人体不同细胞中的转导率,为再生医学和组织工程学研究中报道基因的选择提供依据.方法 将构建的GFP和RFP慢病毒载体分别转染人骨髓间充质干细胞（hMSC）、人脐静脉内皮细胞株（Eahy926）和人肺泡上皮细胞株（A549）.4d后,用流式细胞仪检测GFP和RFP的转导率,并利用激光共聚焦显微镜观察GFP和RFP的表达特征.结果 GFP和RFP在hMSC、Eahy926和A549三种细胞间的转导率均有显著性差异（P﹤0.01）;在hMSC或Eahy926或A549的同种细胞中GFP和RFP的转导率也有显著性差异（P﹤0.01）;RFP在部分Eahy926和A549细胞局部高表达.结论 慢病毒载体介导的GFP和RFP报道基因在人体不同细胞中的转导率明显不同.     

Multicenter clinical evaluation of the new 3rd generation assay for detection of antibodies against hepatitis C virus on the VIDAS® system

BackgroundDetection of antibodies (anti-HCV) against hepatitis C virus (HCV) is indispensable for screening and diagnosis of viral hepatitis and for the viral safety of blood, tissue or organ donations. It gains additional importance by the new HCV drugs which improve the therapeutic possibilities dramatically.ObjectiveTo evaluate the performance of a newly developed immune assay for anti-HCV based on the well-established VIDAS platform.Study designThe assay was evaluated with samples from anti-HCV negative blood donors and from patients with or without HCV markers in six centres in France, Spain and Egypt. The status of the samples was determined by using CE-marked immune assays (Architect, AxSym, Prism, Vitros), two immunoblots (RIBA, Inno-Lia) and/or HCV RNA results.ResultsSpecificity was 99.67% in 10,320 French blood donors without anti-HCV, 99.5% in 200 anti-HCV negative hospitalized European patients and 99.0% in 198 negative patients from Egypt. Sensitivity was 99.7% in 1054 patients pretested positive by other assays; 345 patients with known genotype had genotype 1–6; 61 patients were co-infected with HIV. VIDAS was reactive in 78% of 91 patients with uncertain or very weak anti-HCV. It became on average positive at day 37 with seroconversion panels.ConclusionsThis multicentric, international study with >12,000 samples show that the new VIDAS anti-HCV assay is very suitable for screening and confirmation of HCV infection. Sensitivity, specificity and recognition of seroconversion compare favorably with well-established CE-marked tests and help to clarify discrepant results obtained with other assays.