引物原位标记技术检测SRY基因

目的 建立一种能更有效检测单拷贝基因的引物原位标记技术(primed in situ labeling,PRINS).方法 在传统PRINS技术基础上,引入TsqStatrt抗体、Y染色体上的性别决定区基因(SIBX determining region Y,SRY)4条引物和TSATM Biotin System来检测SRY基因,并以对SRY基因的荧光原位杂交(fluorescence in situ hybridization,FISH)为对照.结果 在PRINS结果中,通过对50个中期分裂相的分析,在Yp11.3的位置上都枪测到特异信号且清晰可辨别,此结果与FISH结果一致且标记信号强度相当.结论 这项改进后的PRINS技术可快速针对单拷贝基因和小DNA片段进行检测.因此,相对于昂贵且费时的FISH技术,是另一种有效的选择.     

应用引物原位标记技术快速检测18三体综合征

目的　探索应用改良的引物原位标记技术快速检测未培养羊水细胞间期核的可能性。方法采用改良的引物原位标记 (primed in situ labeling,PRINS)技术 ,对 2 6 2份羊水细胞中 18号染色体进行分析检测。结果　在未培养羊水细胞间期核和培养细胞的中期分裂相中 ,PRINS技术均能特异性地检测 18号染色体 ,PRINS反应的成功率为 95 %。并发现两个样本为 18三体综合征。结论　引物原位标记技术是一种简便、快速、经济的染色体检测方法 ,具有较强的敏感性和特异性 ,有助于对传统细胞遗传学的分析。     

HER-2/neu基因探针的制备及特异性分析

目的 筛选、制备乳腺癌标志基因--HER-2/neu的标记探针,利用间接荧光原位杂交(FISH)技术,探索其临床应用价值.方法 通过文库构建、多级筛选、PCR鉴定、序列分析等技术获得HER-2/neu基因组片段,缺口转移法制备生物素标记探针,与生物素-亲和素放大系统和多种荧光显色系统组合,针对病理标本、细胞涂片进行间接FISH检测.结果 制备的HER-2/neu基因探针可以特异性地检测乳腺癌阳性、胃癌阳性病理片,也可以特异性检测阳性、阴性细胞标本,并与不同的荧光显色系统结合使杂交信号可以选择性地呈现绿色或红色荧光.结论 高特异性的HER-2/neu基因探针和灵活的显色系统为FISH的临床应用奠定了基础.     

引物介导原位标记技术在检测鼻咽癌石蜡切片HPV-DNA的应用

引物查导原位标记（primed in situ labeling,PRINS)技术是由Koch等在1989年首先提出来的.基本原理是将未标记的寡核苷酸作为引物与细胞或组织中的靶DNA或RNA进行杂交.在该引物的介导和聚合酶的催化下将生物素（地高辛、荧光素等）标记的核苷酸掺入，通过显色，达到原位显示的目的。笔者将此技术用于鼻咽癌石腊切片中人乳头状瘤病毒DNA的检测。     

应用双色荧光原位杂交技术对转基因小鼠进行整合位点的染色体定位

目的 建立一种高度灵敏、特异的双色荧光原位杂交(dual-color fluorescence in situ hybridization,D-FISH)技术,对两种双阳性转基因小鼠外源基因进行整合位点的染色体定位.方法 对一只整合单纯疱疹病毒胸苷嘧啶激酶(herpes simplex virus thymidine kinase,HSV-tk)/增强型绿色荧光蛋白(enhanced green fluorescence protein,eGFP)的转基因小鼠以及两只整合RNA干扰载体(RNA interference,RNAi)的β654地中海贫血模型小鼠进行实验,脾脏细胞经培养后获得中期分裂相标本片,各加入适量生物素、地高辛标记探针混合液杂交,分别用罗丹明红色荧光抗体及FFIE绿色荧光抗体进行D-FISH检测.结果 两种转基因小鼠均能在同一个分裂相上同时检测到双色荧光信号.其中,HSV-tk/eGFP双阳性小鼠的分裂相上出现较强的绿色HSV-tk信号和红色eGFP信号,分别定位于染色体2E5-G3及8A2-A4;β654/RNAi双阳性小鼠检测到红色β654荧光信号及绿色RNAi荧光信号.经定位分析,β654均整合在染色体7D3-E2,RNAi病毒载体则是随机整合,其中一只鼠主要整合在1281位点,而另一只鼠主要整合在染色体1E2.3-1F、3A3两个位点.结论 用自行制备的DNA探针建立了高度灵敏、特异的D-FISH技术,同时结合G显带对双阳性转基因小鼠进行染色体基因定位.该技术平台的建立对于转基因动物和基因治疗动物模型的研究具有非常重要的意义.     

对孕妇外周血中胎儿细胞进行基因检测的三种方法比较

目的　建立用孕妇外周血中胎儿细胞进行基因分析的技术。方法　采用引物延伸预扩增(primed extension preamplification,PEP)法加 PCR、多重引物原位合成技术 (primed in situ labeling,PRINS)和巢式聚合酶链反应 (nested PCR)检测孕妇外周血中单个胎儿细胞的性别决定区 Y基因 (sex- de-termination region Y gene,SRY)基因片段。结果　 PEP加 PCR方法检测 SRY基因的敏感性为 97.39%(14 9/ 15 3) ,特异性为 99.17% (119/ 12 0 )。PRINS技术检测 SRY基因的敏感性为 97.5 6 % (40 / 4 1) ,特异性为10 0 % (35 / 35 )。巢式 PCR检测 SRY基因的敏感性为 80 .0 0 % (2 4 / 30 ) ,特异性为 87.5 0 % (14 / 16 )。结论　PEP加 PCR技术和 PRINS技术是对孕妇外周血中单个胎儿细胞进行基因诊断的可靠技术 ,其敏感性高、特异性强 ,有望成为利用孕妇外周血中单个胎儿细胞进行非创伤产前诊断的常规方法。     

应用双色引物原位标记技术快速检测X和Y染色体

目的发展快速检测染色体的技术，探索应用改良的双色引物原位标记（primed in situlabeling，PRINS）技术快速检测未培养细胞间期核可能性。方法采用改良的双色技术，对205份羊水细胞中X和Y染色体进行分析检测。结果在未培养羊水细胞间期核和培养细胞的中期分裂相中，PRINS技术均能特异性地检测X和Y染色体，PRINS反应的成功率为98％，1个样本检出为47，XXY。结论双色引物原位标记技术是一种快速、简便、经济的染色体检测方法，具有较强的特异性和敏感性，有助于快速诊断染色体畸变。     

介绍一种半套式原位PCR技术

原位聚合酶链反应（原位PCR）是具有聚合酶链反应（PCR）技术的高敏感性和原位来交（IS）肘原位检测能力的新方法。但目前常见的间接式原位PCR技术常因PCR反应过程的非特异扩增而伴有假阳性结果出现,对此我们建立了一种高度特异性的卡套式原位PCR法,并与ISH方法进行了比较。1材料与方法利例石蜡包埋鼻咽癌组织,切片知m,贴于涂有防脱片胶的载玻片L6OoC24h。半套式PCR一对半引物及生物素标记寡核苦酸探针l’l由上海生物工程研究所合成。引物为hcf－2基因主要断裂区外引物A！、内引物AZ及IgV。基因引物JH。半套式原位PCR步…     

应用多色引物原位标记技术在未培养羊水细胞中同时识别18号、X和Y染色体

目的 建立多色引物原位标记(multi-primed in situ labelling)技术在未培养羊水细胞中检测染色体数目异常.方法 用非双膜氧核苷酸阻断的三色引物原位标记技术对未培养羊水细胞中的18、X和Y染色体同时进行标记.结果 成功地在69份未培养羊水细胞间期核中检测到相应的染色体信号,且结果与培养羊水细胞的染色体制备分析及其三色引物原位标记相符合,反应成功率达96%,标记率达85%.结论 该研究方法提高了检测通量,快速、简便、经济可行,值得进一步研究与推广.     

Dystrophin基因缺失检测微阵列构建及应用中技术优化的研究

目的建立Dystrophin基因缺失检测的DNA微阵列技术，并优化其构建及应用中的关键技术条件。方法提取样品基因组DNA，Klenow法随机引物扩增同时FITC荧光标记，并比较生物素标记法。将FITC荧光标记的核酸点样于不同方法活化处理的玻片上并用不同浓度的盐溶液洗脱，测试对DNA固定效率的影响。以分子克隆法获得的Dystrophin基因18个易缺失外显子cDNA片段为探针、APES和poly-Lys联合处理的玻片为基片，制备简易微阵列。分别与DMD/BMD患者及健康人荧光标记的基因组DNA杂交，检测微阵列的质量和评估结果的可靠性。结果APES和poly-Lys联合处理的玻片对核酸固定效率最高，核酸在洗涤过程中的脱落程度随洗液盐浓度的增加而增大；FITC标记步骤简便，生物素标记相对烦琐，但生物素标记杂交后的荧光强度明显高于FITC标记；微阵列杂交信号信噪比较好，各种对照结果满意，检测结果与PCR一致。结论优化了DNA微阵列构建及应用过程中基片表面活化、探针固定、靶基因DNA扩增标记、杂交条件、杂交信号检测分析等方法，为更好地应用微阵列进行Dystrophin基因诊断奠定基础。     

The utility of in situ--based methodologies including in situ polymerase chain reaction for the diagnosis and study of viral infections

Molecular in situ-based assays are a useful adjunct to the diagnosis of viral infections by the surgical and cytopathologist. In some cases, the viral nucleic acids and/or proteins are abundant and easily detected by in situ hybridization or immunohistochemistry. In other cases, such as the one integrated provirus typical of latent retroviral localization, in situ polymerase chain reaction amplification is required to localize the virus in the intact cell. Direct correlation of viral localization and the histologic changes will demonstrate in many cases that routine histopathology often does not provide sufficient information to determine what specific cells are infected and/or the number of infected cells in a given biopsy. By combining this information with, for example, cytokine localization, one can elucidate much about the pathophysiology of the viral infection that cannot be afforded by polymerase chain reaction-based methods alone.     

Modern histochemical methods using enzymes as markers

In the 25 years since the idea that histochemically detectable enzymes could serve as a marker of ligands, the world of histochemistry has undergone a dramatic change. Today slides of immunostained tissue sections may be filed together with hematoxylin- and eosin-stained slides and may be examined at one's leisure. Antigens are now localized at the ultrastructural level as well as at the light microscopic level permitting one to wonder about the intricacies and beauties of living creatures. Genes which mandate our body since the day of conception to the day of death can now easily be probed with enzyme markers. Even with all these success stories, some dreams which I had hoped to accomplish with the method are not yet realized.

Unlike the chemical and physical properties of inert objects, living organisms are provided with a fixed genetic program. By recognizing the point in the program which is being executed, one can deduce the potential activities of the cells and tissues. I hypothesize that this is not an impossible task since (1) histochemistry and immunohistochemistry already provide us information on the activities of cells and tissues, (2) in situ hybridization describes gene activities, and (3) newer methods allow for the determination of potential gene function. Thus it is possible to peek at the future.     

A double labeling technique for performing immunocytochemistry and in situ hybridization in virus infected cell cultures and tissues

This report describes a combined immunocytochemical and in situ hybridization procedure which allows visualization of cellular or viral antigens and viral RNA in the same cell. Cultures infected with visna or measles virus were fixed in periodate-lysine-paraformaldehyde-glutaraldehyde, stained by the avidin-biotin-peroxidase technique using antibodies to viral or cellular proteins and then incubated with radiolabeled specific DNA probes (in situ hybridization). The immunoperoxidase stain was preserved through the hybridization procedure. Nonspecific 'sticking' of probes over peroxidase stained cells was prevented by incorporation of 0.1% Triton X-100 into the hybridization solution and the post-hybridization washes. The in situ hybridization signal (silver grains/cell) on peroxidase-stained cells was reduced relative to hybridization with unstained cells. The double labeling technique was also applied to sections of paraffin-embedded tissues from a sheep infected with visna virus and mice infected with the HNT strain of measles virus. Visna virus RNA was detected in immunocytochemically identified macrophages in the synovium. A greater number of these cells had viral RNA than had viral protein. In measles virus-infected brains viral RNA was detected only in cells with viral protein. This technique provides a new approach to the study of viral pathogenesis by: identifying the types of cells which are infected in the host and identifying points of blockade in the virus life cycle during persistent infections.     

Incidence and distribution of carcinoma in situ in testes removed for germ cell tumour: possible inadequacy of random testicular biopsy in detecting the condition

A total of 127 testicular specimens with germ cell tumours were stained immunohistologically for placental alkaline phosphatase to investigate the presence and topographic distribution of carcinoma in situ cells adjacent to the invasive tumours. Carcinoma in situ was detected in 72% of the cases. In 60% of the cases positive for placental alkaline phosphatase the distribution of carcinoma in situ was not diffuse, as claimed in the literature, but focal. Clinicians screening for carcinoma in situ by only one random biopsy have, therefore, to be aware of possible false negative results.     

Optical,Electrochemical, Magnetic,and Conductive Properties of New Poly(indolocarbazole‐alt‐bithiophene)s

Summary: New alternating copolymers based on indolocarbazole (IC), both two‐ and three‐coupled, and bithiophene (BT) or bis(3,4‐ethylenedioxythiophene) (BiEDOT), were obtained using Stille or Suzuki coupling reactions. Those copolymers have been investigated by cyclic voltammetry, electrochemical quartz crystal microbalance, in situ electron spin resonance, in situ conductivity, and UV‐Vis‐NIR spectroelectrochemistry techniques. All polymer films undergo reversible oxidation processes. One to three isoelectronic oxidation processes, involving one electron per repeat unit, produce radical cations, dications, and radical trications. The oxidative charge is localized in the IC moiety for BT copolymers or over the whole polyconjugated backbone for BiEDOT copolymers. Redox neutral‐polaron conductivities are in the range of 0.02–0.1 S · cm^?1 and polaron‐bipolaron conductivities in the range of 0.1–0.7 S · cm^?1.

Chemical structure of the PIC derivatives.     

Expression of type 1 and P fimbriae in situ and localisation of a uropathogenic Escherichia coli strain in the murine bladder and kidney

Adhesion is an important aspect of bacterial colonisation and induction of human disease. Escherichia coli which infects and causes disease of the urinary tract expresses several adherence factors including type 1 and P fimbriae. Their expression has been implicated in the virulence of E. coli strains infecting the urinary tract, however, the evidence for the expression of these fimbriae in situ has been implied rather than proven. Here we describe in situ detection of E. coli and of fimbrial expression in urinary tract tissue. Kidneys and bladders were isolated from mice infected with the uropathogenic isolate E. coli AD110. The tissue was sectioned and subjected to DNA-rRNA hybridization and indirect immunofluorescent staining with antibodies against type 1 and P fimbriae. Sections of both kidney and bladder stained positive for bacterial cells using a Cy3-labelled E. coli-specific rRNA probe. The same cells in these sections also stained positive for type 1 or P fimbriae using fluorescein-labelled antibodies. Tissue taken from several different time points (2, 6, and 24 hours post infection) showed the presence of bacterial cells which stained positive for fimbrial expression. Bacteria in kidney and bladder sections were observed either as individual cells associated with the mucosa or as members of microcolonies.