FRET在FQ-PCR荧光检测中的应用

简要介绍了荧光共振能量转移（FRET）原理和荧光定量PCR中常用的荧光检测方法．着重讨论了FRET应用于PCR光检测应注意的几个问题，并指出了未来研究趋势。     

荧光漂白恢复、荧光共振能量转移和荧光相关光谱检测的技术特点

荧光漂白恢复（FRAP）、荧光共振能量转移（FRET）和荧光相关光谱（FCS）是三种以荧光为基础的检测技术,常用来研究分子间相互作用。对三种技术的特点做以比较和讨论。     

PAN-Rh6G能量转移荧光猝灭法测定水样中痕量镉(Ⅱ)

目的：根据能量转移荧光猝灭程度,建立一种荧光猝灭法测定环境水样中痕量镉的新方法.方法：在无水乙醇介质中,Cd^2＋与1-（2-吡啶偶氮）-2-萘酚（PAN）形成PAN-Cd^2＋配合物,其吸收光谱与罗丹明6G（Rh6G）的发射光谱有效重叠,发生能量转移,使Rh6G荧光猝灭,从而建立了痕量镉的荧光猝灭测定新方法.结果：在优化实验条件下,在31～800 ng/ml浓度范围内,Rh6G荧光猝灭程度与PCd^2＋呈现良好的线性关系（r=0.9999）.方法的检出限为9.0 ng/ml.结论：方法灵敏、快速、简便,用于环境水样中痕量镉的测定,结果满意.     

量子点荧光探针在生物医学中的应用进展

量子点（半导体纳米微晶体）作为一种新型荧光探针,在生物医学领域中应用已引起国内外科学工作者的极大关注。文章主要概括了量子点优于传统荧光染料的特性、量子点荧光探针的生物标记方式及其在活细胞荧光标记及组织光学成像、肿瘤细胞示踪及检测、荧光免疫分析和微生物学等方面的应用,并对其在兽药多残留检测的发展前景进行了展望。     

荧光钙离子测定技术在医学研究中的意义及应用

荧光钙离子测定技术日臻成熟,已成为研究细胞学机制的重要手段.本文对其在医学研究中 的应用和意义进行讨论并介绍开展此技术的若干体会.     

杂交探针技术结合熔解曲线快速鉴别水痘一带状疱疹病毒野毒株／疫苗株

目的利用杂交探针结合熔解曲线分析技术,建立一种快速、可靠的适用于鉴别水痘一带状疱疹病毒（Varicella—zosterVirus,VZV）野毒株与疫苗株的方法。方法采集临床诊断为水痘患者的水疱液并进行病毒分离,抽提阳性分离物和部分阳性水疱液的基因组脱氧核糖核酸（DeoxyribonucleicAcid,DNA）。以荧光素Cy5．5标记检测探针5端,相应的锚定探针在3端用FAM羧基荧光索标记,再从研究对象基因组DNA中用聚合酶链反应（PolymeraseChainReac—tion,PCR）特异引物扩增ORF62基因中301碱基对（basepair,bp）片段,将其与特异探针杂交后,引发荧光共振能量转移,通过罗氏LightCycler实时PCR仪描绘扩增的目标DNA片段的熔解曲线,根据熔解温度（MeltingTemperature,Tm）峰值对待测标本进行鉴别。最后,用限制性片段长度多态性（RestrictionFragmentLengthPolymorphism,RFLP）方法验证杂交探针结合熔解曲线鉴别野毒株／疫苗株的准确性。结果VZVOka疫苗株的扩增产物与检测探针的Tm为52．47℃,而水痘原始临床标本／临床分离株与检测探针的Tm在57．33℃～60．50℃。通过检测DNA扩增产物与VzVORF62探针Tm的差异可以明确鉴别VZV野毒株和疫苗株。利用杂交探针技术结合熔解曲线的方法对水痘原始临床标本及分离株进行野毒株／疫苗株的鉴别结果与传统的RFLP方法进行鉴别的结果完全一致。结论杂交探针技术结合熔解曲线操作简便省时,不易交叉污染;获得的病毒野毒株／疫苗株特征性强,适用于VZV野毒株／疫苗株在临床和流行病学调查中的快速鉴别诊断。     

061固相细胞计数法--一种快速检测单细胞细菌的食品微生物新检测方法

固相细胞计数(SPC)是一项新技术,可在单细胞水平快速检测细菌而不需要生长相.滤过样品后,存留的微生物在滤膜上进行荧光标记,采用激光扫描设备自动计数.每个荧光点可直观地由通过计算机驱动的流动台连接到ChemScan上的落射荧光显微镜来检测.根据所使用的荧光标记,在几小时内可获得有关微生物特性及生理状态的信息.尽管SPC最初建议用于测定水中及其他液体样品中活细菌总数,但如果可以从不能滤过的基质中分离出细菌,它将是检测及计数食物样品中细菌的有效技术.尤其对于生长缓慢的微生物,检测用时短使该方法明显优于传统平板计数法.本文讨论了SPC的基本原理以及检测副结核分枝杆菌(My-cobacterium paratuberculosis)的可能性,后者是牛奶中生长缓慢的细菌的代表菌.     

FISH技术在产前诊断胎儿染色体数异常中的应用

目的 探讨荧光原位杂交(FISH)技术在快速产前诊断胎儿染色体非整倍体异常中的价值.方法 使用荧光原位杂交技术,选用荧光素标记的双色13/21染色体位点特异性探针和三色18/X/Y染色体着丝粒探针,检测760例胎儿羊水细胞.结果 采用双色13/21号和三色18/X/Y染色体荧光探针检测间期未培养羊水细胞,发现8例21三体综合征,1例13三体综合征,1例45,XO,1例47,XXX,3例性染色体嵌合体.荧光原位杂交检测结果 和常规细胞遗传学检测结果 相比,两者符合率为99%.结论 荧光原位杂交技术在产前快速诊断胎儿染色体非整倍体异常有很高的临床价值.     

母血中胎儿细胞的研究现状及展望

母血中主要存在三种胎儿细胞，即滋养层细胞、胎儿白细胞及胎儿红细胞。其中胎儿有核红细胞被认为最有前途的可用于进行产前诊断的胎儿细胞。目前富集和分离胎儿细胞的方法主要有荧光激活细胞分离技术和磁性细胞分离技术两种。利用母血中的胎儿细胞可进行胎儿性别预测和一些遗传性疾病的产前诊断。研究表明，母血中胎儿细胞的数量与一些妊娠并发症和产后自身免疫性疾病有关。     

8-羟基脱氧鸟苷与鱼精蛋白相互作用的研究及其分析应用

目的建立一种简便的8-OH-dG检测新方法.方法在pH 8.6的Tris-HCI缓冲液中,8-OH-dG阴离子与鱼精蛋白阳离子作用,形成静态复合物,导致鱼精蛋白的荧光强度减弱.结果根据F(o)rster非辐射能量转移理论,鱼精蛋白与8-OH-dG结合常数为KA=3.10×104 L/mol(t=25℃).确定供体-受体之间的距离r=3.47 nm和能量转移效率E=0.24.实验表明,8-羟基脱氧鸟苷与鱼精蛋白的作用为静态荧光猝灭过程.方法的检测下限为0.18 μg/ml,回归方程为(y)=8.7+70X(μg/ml),r=0.9967.结论该方法快速简便,已成功用于加标样品的分析.     

Fluorescence spectroscopic investigation of the interaction between chloramphenicol and lysozyme

The interactions between chloramphenicol and lysozyme were studied using fluorescence, UV/vis and circular dichroism spectra. The results proved the mechanism of fluorescence quenching of lysozyme by chloramphenicol is due to the formation of lysozyme–chloramphenicol complex. The thermodynamic parameters, enthalpy change (ΔH) and entropy change (ΔS) for the reaction, were calculated to be −12.41 kJ mol⁻¹ and 37.99 J mol⁻¹ K⁻¹, which indicated that hydrophobic force and hydrogen bond were the dominant intermolecular forces in stabilizing the complex. The distance r = 3.99 nm between donor and acceptor was obtained according to Förster's theory. In addition, the alterations of lysozyme secondary structure in the presence of chloramphenicol were confirmed by the evidences from circular dichroism, synchronous and three-dimensional fluorescence spectroscopy.     

激光扫描共聚焦显微技术及其在神经、肿瘤相关研究中的应用

介绍了近年来新出现的共聚焦显微镜新的种类,如传统激光共聚焦和活细胞激光共聚焦,对其功能及在神经、肿瘤研究中的应用进行了综述,使现代显微镜能够更加深入研究和分析细胞的变化过程和结构。活细胞激光扫描共聚焦显微镜与双光子激光扫描共聚焦显微镜实现了对肿瘤、神经活细胞长时间地动态观测,可更加真实地揭示细胞凋亡的机制与规律。     

High linear-energy-transfer radiation can overcome radioresistance of glioma stem-like cells to low linear-energy-transfer radiation

Ionizing radiation is applied as the standard treatment for glioblastoma multiforme (GBM). However, radiotherapy remains merely palliative, not curative, because of the existence of glioma stem cells (GSCs), which are regarded as highly radioresistant to low linear-energy-transfer (LET) photons. Here we analyzed whether or not high-LET particles can overcome the radioresistance of GSCs. Glioma stem-like cells (GSLCs) were induced from the GBM cell line A172 in stem cell culture medium. The phenotypes of GSLCs and wild-type cells were confirmed using stem cell markers. These cells were irradiated with ⁶⁰Co gamma rays or reactor neutron beams. Under neutron-beam irradiation, high-LET proton particles can be produced through elastic scattering or nitrogen capture reaction. Radiosensitivity was assessed by a colony-forming assay, and the DNA double-strand breaks (DSBs) were assessed by a histone gamma-H2AX focus detection assay. In stem cell culture medium, GSLCs could form neurosphere-like cells and express neural stem cell markers (Sox2 and Musashi) abundantly in comparison with their parental cells. GSLCs were significantly more radioresistant to gamma rays than their parental cells, but neutron beams overcame this resistance. There were significantly fewer gamma-H2AX foci in the A172 GSLCs 24 h after irradiation with gamma rays than in their parental cultured cells, while there was no apparent difference following neutron-beam irradiation. High-LET radiation can overcome the radioresistance of GSLCs by producing unrepairable DNA DSBs. High-LET radiation therapy might have the potential to overcome GBM''s resistance to X-rays in a clinical setting.     

Nucleic acid immunization: a prophylactic gene therapy?

Nucleic acid (NA) vaccines may offer the safety of subunit or inactivated vaccines and, at the same time, provide the advantages of live recombinant vaccines, such as induction of a protective cellular immune response. In Germany, the so-called ‘Gene Law’ regulates the genetic modification of organism such as prokaryotic or eukaryotic cells for the construction of recombinant NAs intended for use as NA vaccines. Neither NAs nor human being treated with NAs are subject to Gene Law regulations but preclinical laboratory experiments are regulated by the Gene Law. Gene therapy, as defined in a recent draft of a European guideline for the production of gene therapeutics, includes the genetic modification of human somatic cells via transfer of NAs and thus includes NA vaccines. The guideline provides recommendations for the production of NA vaccine for human use and for preclinical safety testing. NA vaccines are products derived by biotechnological processes, as defined in part A of the annex of Council Regulation (EEC) No. 2309/93 of 22 July 1993. Applications for marketing authorization in Member States of the European Union will thus be reviewed by the European Agency for the Evaluation of Medicinal Products starting from 1 January 1995. Inoculation NAs encompassing a full-length but int/nef-defective simian immunodeficiency provirus allowing limited replication of viruses released is being investigated at the Paul-Ehrlich-Institute as a model for a NA vaccine against AIDS. The system may offer a promising way towards a vaccine and will also be used to study safety requirements for future human use of NA vaccines.     

Human cell responses to ionizing radiation are differentially affected by the expressed connexins

In multicellular organisms, intercellular communication is essential for homeostatic functions and has a major role in tissue responses to stress. Here, we describe the effects of expression of different connexins, which form gap junction channels with different permeabilities, on the responses of human cells to ionizing radiation. Exposure of confluent HeLa cell cultures to ¹³⁷Cs γ rays, 3.7 MeV α particles, 1000 MeV protons or 1000 MeV/u iron ions resulted in distinct effects when the cells expressed gap junction channels composed of either connexin26 (Cx26) or connexin32 (Cx32). Irradiated HeLa cells expressing Cx26 generally showed decreased clonogenic survival and reduced metabolic activity relative to parental cells lacking gap junction communication. In contrast, irradiated HeLa cells expressing Cx32 generally showed enhanced survival and greater metabolic activity relative to the control cells. The effects on clonogenic survival correlated more strongly with effects on metabolic activity than with DNA damage as assessed by micronucleus formation. The data also showed that the ability of a connexin to affect clonogenic survival following ionizing radiation can depend on the specific type of radiation. Together, these findings show that specific types of connexin channels are targets that may be exploited to enhance radiotherapeutic efficacy and to formulate countermeasures to the harmful effects of specific types of ionizing radiation.     

实时荧光定量PCR技术进展

实时荧光定量PCR(real_time quantitative PCR)技术是一种新型的核酸定量检测、分析技术,它通过在PCR扩增反应过程中加入荧光物质,使得对反应过程的实时监控成为可能。它具有实时监测、定量准确、灵敏度高、反应速度快、重复性好及PCR反应后不需电泳检测等优点,已逐步成为分子生物学研究中的重要工具。     

Recombinant West Nile virus envelope protein E and domain III expressed in insect larvae protects mice against West Nile disease

In this study, West Nile virus (WNV) envelope (rE) protein and its domain III (rDIII) were efficiently expressed in a cost-effective system based on insect larvae as non-fermentative living biofactories. Mice immunized with the partially purified rE or rDIII elicited high antibodies titers that neutralized viral infectivity in cell culture and in suckling mice. All vaccinated animals were fully protected when challenged with neurovirulent WNV NY99. Passive transfer of protective antibodies from immunized mothers to their offspring occurred both by transplacental and lactation routes. These results indicate that the insect-derived antigens tested may constitute potential vaccine candidates to be further evaluated.