原位PCR对胃癌和胃炎组织内幽门螺旋杆菌的检测

为探讨幽门螺旋杆菌(Helicobacter pylori, Hpy)与胃癌发病的关系,我们建立了原位(PCR(in situ PCR,ISPCR)方法,并使用它对胃炎和胃癌组织内的幽门螺旋杆菌进行了检测。发现在胃炎组织内Hpy的感染率为83.3％,胃癌组织内的感染率为65％。在11例胃癌阳性标本中,4例为细胞核阳性,2例细胞浆阳性,5例为所带正常胃腺组织内阳性。提示,Hpy的致癌机理,除与细菌的长期刺激外,尚可能与其参与细胞的功能调控有关。     

Optimized beta-galactosidase staining method for simultaneous detection of endogenous gene expression in early mouse embryos

beta-Galactosidase (beta-gal) is one of the popular reporters for detecting the expression of endogenous or exogenous genes. Here we report 6-chloro-3-indoxyl-beta-D-galactopyranoside (S-gal) is more sensitive for beta-gal activity than 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-gal), particularly during the early developmental stages of mouse embryos. Further, we successfully combined beta-gal staining with S-gal and in situ hybridization using DIG-labeled probes in both whole and sections of early stage embryos due to the sensitivity and color compatibility of S-gal.     

mecA基因PCR扩增法检测耐甲氧西林金黄色葡萄球菌

目的 应用mecA基因PCR扩增法检测耐甲氧西林金黄色葡萄球菌（methicillin resistant staphylococcus aureus,MRSA）。方法 临床分离的70株金黄色葡萄球菌,应用mecA基因PCR扩增法鉴定MRSA,并与苯唑西林纸片扩散法进行比较。结果 70株金黄色葡萄球菌用PCR扩增法和纸片扩散法有6株鉴定有差异,4株。mecA基因阳性而纸片扩散法鉴定为敏感,1株mecA基因阳性纸片扩散法鉴定为临界耐药,1株mecA基因阴性却表现为苯唑西林耐药,2种方法符合率为91．43％。结论 mecA基因PCR扩增法可以准确、快速判定MRSA,特别是对隐匿型或低水平耐药菌株的检出有重要的价值。     

逆转录病毒载体在血友病A基因治疗中的研究进展

基因治疗是彻底治愈血友病A的最理想方法,逆转录病毒是最为常用的载体之一,本文对逆转录病毒在血友病A基因治疗中的研究进展作一综述。     

Adenoviral gene transfer to the neonatal rat pulmonary circulation

BACKGROUND: Gene delivery to the pulmonary circulation has been studied in adult animals, but has not been extensively investigated in neonates. METHODS: We tested the ability of recombinant, replication-defective adenovirus to transduce the pulmonary circulation when delivered by percutaneous ventricular puncture. Five-day-old rat pups were injected with 10(7) to 10(10) particles (approximately 10(5) to 10(8) pfu) in 30 micro l total volume. RESULTS: Using RT-PCR, we detected transgene expression in both lung and liver at all dosages. However, whereas only 1/6 pups injected with 10(7) particles had detectable expression, 8/9 pups in the two highest dose groups had detectable expression. In the highest dose group expression was approximately 5-fold greater in lung than liver, though in the lower dose groups no difference between lung and liver was found. Expression decreased by only 25% from day 4 through the last time point at day 28 in lung, whereas liver expression was undetectable in 7 of 9 samples on day 28. Histopathological examination demonstrated expression both within the media of large arteries and in small, peripheral arteries and capillaries, with a concentration of expression in the most distal areas of both the lungs and liver. No evidence of inflammation was seen. CONCLUSIONS: We conclude that the neonatal pulmonary circulation can be effectively transduced using systemic adenoviral vector injection, has more sustained expression than liver, and may be a target for therapeutic gene delivery.     

Application of groEL gene for the species-specific detection of Vibrio parahaemolyticus by PCR

Aims: Vibrio parahaemolyticus is a significant cause of human gastrointestinal disorders and is transmitted through ingestion of raw or undercooked contaminated seafood. We used the groEL gene for the species‐specific detection of V. parahaemolyticus from artificially inoculated shellfish, fish and seawater. Methods and Results: The nucleotide sequences of 24 Vibrio and seven non‐Vibrio spp. were compared, and less conserved regions were selected for the designing of primer sets. To detect V. parahaemolyticus specifically, PCR conditions were standardized and tested to evaluate the specificity of primers. A 510‐bp band was appeared only from V. parahaemolyticus by PCR. Notably, the detection was shown to be functional at high annealing temperature above 68°C. The groEL primers detected 100 pg and 1 ng of DNA purified from V. parahaemolyticus culture and artificially infected oyster tissue, respectively. Conclusions: The groEL gene is a potential marker for the species‐specific detection of V. parahaemolyticus and could be used to detect this bacterium in contaminated food by PCR. Significance and Impact of the Study: PCR using primers designed from groEL gene provide an efficient method for the accurate identification of V. parahaemolyticus from contaminated samples.     

Anisakis pegreffii: a quantitative fluorescence PCR assay for detection in situ

To facilitate improved diagnosis and detection of the third stage larva (L3) of Anisakis pegreffii from the Minnan-Taiwan bank fishing ground in Taiwan Strait, a real-time PCR method for the detection in situ and differentiation was developed to amplify a region of the second internal transcribed spacer (ITS-2) of this parasite. The real-time PCR assay was capable of detecting 1/3 of a single L3 in 30 mg of marine fish tissue, and also exhibited a high level of specificity for A. pegreffii, no fluorescence signals were observed in other five major larval anisakid species found in commercial marine fishes caught in this fishing ground.     

基因治疗研究中脂质体介导的基因转移技术

对于脂质体的深入研究特别是阳离子脂质体的研制使其逐步成为重要的基因转移载体之一,并且初步应用于基因治疗研究,同时多种靶向脂质体的研制也为体内靶向基因转移和表达奠定了基础。本文就脂质体的结构、功能、在基因治疗研究中的应用以及各种靶向脂质体的研制进行了介绍。     

植物染色体原位杂交技术的发展与现状

本文主要介绍植物染色体原位杂交技术的发展历史,评述适用于不同研究目的的各种主要原位杂交技术的基本原理和方法,并介绍该技术在植物细胞遗传学领域的应用和发展。 Abstract：In this paper,we briefly introduce the development of in situ hybridization of plant chromosome. The fundamental principle and method of many main kinds of in situ hybridization have been reviewed for different research purposes,and their application and development on plant cytogenetics also been recommended.     

Optimized lipopolyplex formulations for gene transfer to human colon carcinoma cells under in vitro conditions

BACKGROUND: Polycation (PC, polyplex), cationic lipid (CL, lipoplex), and a combination of PC/CL (lipopolyplex) formulations were investigated for gene transfer to slow-proliferating human colon carcinoma cell lines (COGA). METHODS: The luciferase reporter gene was complexed with either PC, CL, or PC/CL. PCs included linear (PEI22lin, 22 kDa) and branched polyethylenimine (PEI2k, 2 kDa; PEI25br, 25 kDa) and poly-L-lysine (PLL18 with 18 lysine monomers). CLs included DOCSPER, DOSPER and DOTAP. Lipopolyplexes were formed by either sequentially first mixing DNA with PC or CL, followed by addition of CL or PC, respectively, or simultaneously with both PC and CL. Particle size and zeta-potential were determined and gene transfer and cytotoxicity were quantified on COGA-3, -5, -12, HeLa and Sw480 cells. RESULTS: The highest gene transfer was achieved when DNA was first complexed with PC followed by CL. At low ionic strength, particles were small (50-130 nm) with a zeta-potential of +20-40 mV. At physiological ionic strength, only lipoplexes of DOCSPER or DOSPER and their respective lipopolyplexes with PEI25br were stable to aggregation (140-220 nm). Lipopolyplexes of PEI25br were between 5- to 400-fold more efficient compared to the corresponding lipoplexes or polyplexes in all cases. Chloroquine did not significantly affect lipopolyplex-mediated gene transfer. CONCLUSIONS: Lipopolyplex formulations of PEI25br in combination with multivalent CLs (DOCSPER, DOSPER) are promising tools for in vitro and potentially also in vivo gene transfer to colorectal cancer cells.     

Localization by in situ hybridization of a low copy chimaeric resistance gene introduced into plants by direct gene transfer

Summary An in situ hybridization method was developed for detecting single or low copy number genes in metaphase chromosomes of plants. Using as a probe ³H-labelled plasmid pABDI, which confers kanamycin resistance (Km^r) to transformed cells. DNA introduced into the plant genome by direct gene transfer was detected with a high efficiency: about 60% to 80% of interphase and metaphase plates showed a strong signal. The insertion site of the Km^r gene in two independent transformants was localised on different homologous chromosome pairs. This result independently confirmed previous genetic data which had indicated that transformed DNA was integrated into plant chromosomes in single blocks.     

全细胞多重PCR检测蓝藻、微囊藻及产毒微囊藻方法初探

选取三对分别针对微囊藻、蓝藻16S rDNA及微囊藻毒素合成酶基因mcyB的保守序列的特异性引物209F/409R、27F1/409R、MTR/MTF,其中409R为一条共用引物。设计并优化了一种可以同时检测蓝藻和微囊藻的两重全细胞PCR方法和一种可以同时检测蓝藻、微囊藻和可产毒微囊藻的三重全细胞PCR方法,并且测试了这两种PCR反应的灵敏度区间,分别为10⁵～10³cell·mL^-1、10⁵～10²cell·mL^-1。对采集水库水样检测结果表明双重全细胞PCR方法可以直接应用于对天然水样的检测,三重全细胞PCR方法可用于实验室培养藻细胞的筛查。全细胞多重PCR方法具有快速、简便、准确等特点,在水体微囊藻毒素检测预警方面具有应用价值。     

原位PCR和原位杂交检测蛋鸡J亚群禽白血病病毒

根据ALV_J原型株HPRS10 3株gp85基因的内部序列 ,和pol基因的 3′端设计一对引物H5 H7。从发生ML病死蛋用型鸡的肿瘤、骨髓、肝脏、脾脏和输卵管组织中提取总RNA ,反转录为cDNA ,经PCR扩增得到长度为 5 4 5bp的ALV_JcDNA特异性探针。探针定位于 5 2 5 8～ 5 80 2bp。将病鸡的组织石蜡切片置HybaidExpress原位PCR仪平台上 ,以H5 H7为引物进行原位PCR扩增。应用地高辛标记的cDNA探针对原位PCR扩增后切片进行了原位杂交检测。结果在待检组织肿瘤组织、十二指肠、骨髓中出现明显的阳性信号。睾丸、肺、胰腺、大脑、输卵管、肾脏均检出散在的阳性信号。这是国内外首次从分子水平证明蛋鸡J亚群禽白血病。     

四种瘿蜂体内Wolbachia感染的PCR检测及感染株系的wsp基因序列分析

Wolbachia为节肢动物等的细胞质共生细菌,能对宿主的繁殖模式进行调控,包括诱导胞质不亲和、孤雌生殖、雌性化及雄性致死。本文采集了分布于美国的4种瘿蜂,利用Wolbachia的wsp基因特异性引物,对其Wolbachia的感染进行了PCR检测,证实了栎结瘤瘿蜂Callirhytis punctata Bassett和摇鼓栎瘿蜂Dryocosmus palustris Osten Sacken体内具Wolbachia共生,感染率分别为60%和36%。栎结瘤瘿蜂和摇鼓栎瘿蜂Wolbachia的wsp基因序列长度分别为564 bp和561 bp。栎结瘤瘿蜂与摇鼓栎瘿蜂Wolbachia的wsp基因序列的一致性为94%。栎结瘤瘿蜂与同为栎瘿蜂族的Andricus solitarius(strain 1)和Neuroterus macropterus,及客瘿蜂族的Synergus crassicorni的Wolbachia的wsp基因序列完全一致,与其他瘿蜂Wolbachia的wsp基因的序列一致性介于79%⁹9%之间。在NJ系统发育树中,栎结瘤瘿蜂与栎瘿蜂族的A.solitaries(strain 1),N.macropterus和B.pallida,以及客瘿蜂族的S.crassicornis的Wolbachia同属一分支,而摇鼓栎瘿蜂与栎瘿蜂族的麦氏安瘿蜂的Wolbachia聚集在同一分支。除客瘿蜂族的Ceroptres cerri感染的Wolbachia属于B群之外,其他瘿蜂感染的Wolbachia均属于A群。此外,本文采集的栎结瘤瘿蜂和摇鼓栎瘿蜂营有性生殖,说明Wolbachia的共生并不诱导其营产雌孤雌生殖。     

Rapid and simultaneous detection of chromosome Y- and 1-bearing porcine spermatozoa by fluorescence in situ hybridization

This study was carried out to develop a rapid and simultaneous detection system of chromosome Y- and 1-bearing porcine spermatozoa by fluorescence in situ hybridization (FISH). Chromosome Y- and 1-specific DNA probes were produced by polymerase chain reaction with digoxigenin (Dig)- or biotin-dUTP. The hybridization probe mixture of labeled Y-chromosome and chromosome 1-specific DNA was applied to the preparation, immediately denatured at 75°C for 8 min, hybridized for 5 min at 37°C and overall FISH steps were done within a few hours. When double FISH with Dig-labeled chromosome Y-specific and biotin-labeled chromosome 1-specific probes was applied to sperm nuclei pretreated with dithiothreitol, the average of 50.9% of sperm nuclei had the Dig-signal, 99.2% of the sperm nuclei had the biotin-signal and the average of 0.3% of sperm nuclei showed no signal. The putative rate of Y-bearing spermatozoa ranged from 49.8% to 52.8% among 5 boars and the average putative rate of Y-bearing spermatozoa was 51.0%. The results indicated that a rapid and simultaneous FISH with chromosome Y- and 1-specific porcine DNA probes produced by PCR made possible more accurate assessment of Y-bearing porcine spermatozoa. © 1996 Wiley-Liss, Inc.     

Somatic gene transfer into the lactating ovine mammary gland

Background

Somatic gene therapy requires safe and efficient techniques for the gene transfer procedure. The ovine mammary gland is described as a model system for the evaluation of somatic gene transfer methods.

Methods

Different gene delivery formulations were retrogradely injected into the mammary gland of lactating sheep. The efficiency of the gene transfer was subsequently measured by the detection of the secreted transgene products in the milk. To counteract the milk flow in the lactating gland caused by the permanent milk production, a newly developed pretreatment of the mammary gland with hyperosmotic solutions was applied. In addition, in vivo electroporation of DNA into the mammary gland is described.

Results

Gene transfer using naked DNA or simple complexes of DNA with polycations did not result in traceable amounts of reporter gene products. However, utilizing the complex cationic lipid DOSPER, a peak expression of about 400 ng/ml was observed 6 days after transfection. Maximum expression rates of more than 1 µg/ml were obtained by combining hyperosmotic pretreatment and receptor‐mediated gene transfer. For the in vivo electroporation, the proof of principle for this technique in the mammary gland is reported.

Conclusions

The ovine mammary gland turned out to be a very well suited as a model system for evaluation and optimization of various gene transfer protocols. Copyright © 2002 John Wiley & Sons, Ltd.