SPECIFIC IDENTIFICATION OF HERPES SIMPLEX VIRUS IN HUMAN ESOPHAGUS WITH RAPID IN SITU HYBRIDIZATION IN 5 CASES

HUMAN herpes simplex virus esophagitis （HSVE） was first reported in 1940 by Johnson. ^1HSVE usually occurs in immunocompromised patients,such as those with acquired immunodeficiency syndrome （AIDS）, 2-4 malignancies, cutaneous burns, connective tissue diseases, inflammatory bowel disease, those taking immuno-suppressive therapy, and those undergoing organ transplantation,5 etc. In the immunocompetent individuals, HSVE is rare, having been reported in 39 cases and mainly affecting young males^6,7 The aim of this study was to delineate the clinical experience in the diagnosis of HSVE using rapid in situ hybridization and assess the various detection methods.

Identification of Prorocentrum minimum and Takayama pulchella by fluorescence in situ hybridization through epifluorescence microscopy and flow cytometry

Partial rDNA sequences of Prorocentrum minimum and Takayama pulchella were amplified, cloned and sequenced, and these sequence data were deposited in the GenBank. Eight oligonucleotide probes （DNA probes） were designed based on the sequence analysis. The probes were employed to detect and identify P. minimum and T. pulchella in unialgal and mixed algal samples with a fluorescence in situ hybridization method using flow cytometry. Epifluorescence micrographs showed that these specific probes labeled with fluorescein isothiocyanate entered the algal cells and bound to target sequences, and the fluorescence signal resulting from whole-cell hybridization varied from probe to probe. These DNA probes and the hybridization protocol we developed were specific and effective for P. minimum and T. pulchella, without any specific binding to other algal species. The hybridization efficiency of different probes specific to P. minimum was in the order： PM18S02 PM28S02 〉 PM28S01 〉PM18S01, and that of the probes specific to T. pulcheUa was TP18S02 TP28S01 〉 TP28S02 〉TP18S01. The different hybridization efficiency of the DNA probes could also be shown in the fluorescent signals between the labeled and unlabeled cells demonstrated using flow cytometry. The DNA probes PM18S02, PM28S02, TP18S02 and TP28S01, and the protocol, were also useful for the detection of Mgae in natural samples.

Differential expression of autocrine motility factor receptor (AMFR) mRNA in normal and cancer cells detected by in situ hybridization

The receptor for autocrine motility factor (AMFR) is known to be involved in the process of AMF-mediated cell migration and metastasis. This paper describes the procedures of non-radioactive in situ hybridization (ISH) detection of AMFR mRNA in both paraffin-embedded surgical sections and cultured cells using either biotinylated oligonucleotide probes or digoxigenin-labeled RNA probes. The results showed that the AMFR mRNA was expressed at an enhanced level in hyperplaJstic and malignant tissues of breast and prostate cancer patient surgical specimens, indicating that the elevated AMFR expression was associated with the tissue malignancy Moreover, AMFR mRNA was detected in both normal and earcinoma cells when cultured at a subconfluent density. However, AMFR expression was inhibited in confluent normal (3T3-A31 murine fibroblast and FHs738BL human bladder) cells while it continued to express in carcinoma (J82 human bladder)and metastatic (3T3-M murine fibroblast) cells irrespective of cell density This suggested a cell-cell contact downregulation of AMFR mRNA expression in normal but not in cancer cells. The ISH data obtained in this study are closely consistent with the AMFR protein expression pattern previously reported, implying that the differential expression of AMFR gene may be regulated and controlled at the transcriptional level.

Application of dual color fluorescence in situ hybridiza tion (D-FISH) to the diagnosis of a 49, XXXXY chromo somal abnormality

To study the technique of D-FISH and its application in the diagnosis of a 49, XXXXY chromosomal abnormality. Methods: Biotin-labeled alpha satellite X chromosome DNA (pBamX7) probe and digoxi-genin-labeled Y chromosome long arm terminal repetitive sequence (pY3.4) probe in situ hybridized with pre-treated slides of peripheral blood chromosome and interphase nucleus. After washing, the slides were treated with avidin-FITC, rhodamine-FITC and anti-avidin, amplified with an additional layer and counter-stained with DAPI in an antifade solution. The hy bridization signals and chromosomal or interphase nucleus settings were observed respectively with WIB, WIG and WU filters under fluorescent microscope (Olympus AX-70) and the number of metaphase chromosome and interphase nucleus in the peripheral blood was counted. Results: The biotin-labeled pBamX7 probe showed 4 green hybridization signal and the digoxigenin-labeled pY3.4 probe showed 1 red hybridization signal. The chromosome or cytoplasm counter-stained with DAPI showed blue. The positive rate of X chromosome hybridization signal for the 350 metaphase chromosomes and interphase nucleus was 91.43 % and 92. 57 %, respectively, while that of the Y chromosome hybridization signal was 99.5 % and 99.8 %, respectively. Conclusion: D-FISH is a valuable technique in diagnosing 49, XXXXY chromosomal abnormality and other sex chromosomal abnormalities. [Reprod Contracep (in Chinese) 2002; 22: 287]

Preparation of Digoxigenin labelled Probe and Detection of HBV DNA in Liver and Extrabepatic Tissue with in Situ Hybridization

A new rapid technique for intrahepatic and extrahepatic HBV DNA detection by using digoxigenin labelled probe with in situ hybridization was developed.This technique has the advantage of being non-radioactive and a quick procedure yielding stable results and showing a clear background.

生物素标记的重复DNA序列与黑麦染色体的原位杂交

本研究以两个黑麦重复DNA序列pSc119.1和pSc119.2作探针进行原位杂交,研究其在小麦和黑麦染色体上的分布及在检测黑麦染色质中的应用。实验结果表明,pSc119.1分布于所有黑麦染色体的长短臂上,但在小麦染色体上几乎检测不到杂交信号,证明 pSc119.1对黑麦染色体具有专化性。进一步用该探针与小麦品种“Amigo”的体细胞染色体进行原位杂交,可明显检测出其中一对含IRS的染色体。 pSc119.2则主要分布在黑麦染色体的端粒区,但在其它部位也可看到一些弱的杂交位点。另外, pSc119.2也能在小麦染色体上产生杂交带型。

鸡包涵体肝炎病毒DNA探针的制备及其原位杂交的应用

用限制酶HindⅢ和Ecorl双切鸡包涵体肝炎六邻体蛋白基因片段重组质粒 ,得到大小 6 36bp的基因片段 ,用地高辛标记制备DNA探针 ,其灵敏度为 0 .1～ 0 .3ng/ μL。用该探针对感染鸡包涵体肝炎病毒的雏鸡肝组织进行原位杂交 ,结果表明病毒经口感染后 12h肝细胞内出现病毒的复制 ,3～ 7d时病毒复制明显 ,9～ 16d病毒复制减弱。原位杂交表明鸡包涵体肝炎病毒主要定位于核内 ,同时也可进入胞浆中。地高辛标记鸡包涵体肝炎病毒DNA探针对检测该病毒DNA的灵敏度高 ,特异性强 ,操作方便 ,该探针可用于本病的分子病理学研究和特异性诊断。

DNA探针鉴定转基因鸡早期性别

质粒ｐＵＧＤ１２０１经扩增、提取、纯化、酶切等一系列过程而获得了Ｗ染色体特异性ＤＮＡ片段，继而获得用地高辛标记的Ｗ染色体ＤＮＡ探针。用该探针通过原位杂交法对鸡Ｘ期胚盘细胞成功地进行了性别鉴定。

线粒体DNA与人细胞癌变的关系——癌细胞核基因组mtDNA探针FISH分析

探讨mtDNA与细胞癌变的关系。方法 :采用PCR方法制备地高辛标记的 3条人mtDNA探针 ,对 6株人癌细胞系和 1例原代培养人皮肤成纤维细胞的染色体或间期细胞核进行荧光原位杂交分析。结果 :3条mtDNA探针在 6个癌细胞系的部分染色体或间期核中均存在阳性杂交信号 ,而在成纤维细胞核中未出现阳性杂交信号。结论 :表明癌细胞核基因组中存在mtDNA的同源序列 。

肝细胞内HBV DNA原位杂交的研究

应用生物素标记HBV DNA(乙肝病毒脱氧核糖核酸)作探针,对129例肝病患者肝组织作原位杂交研究。HBV DNA在慢性活动性肝炎中检出率最高(80.7％),显著高于肝硬化、慢性小叶性肝炎、急性肝炎及原发性肝癌组。具有乙肝复制指标阳性患者的肝细胞内HBV DNA检出率(82.0％)明显高于非复制组(63.0％),P<0.05。本研究观察到;HBB DNA阳性肝细胞与肝细胞坏死灶关系密切,多位于肝细胞坏死灶中间或(和)周边,其中以局灶型分布的HBVDNA阳性肝细胞与肝细胞坏死灶关系密切。提示HBV复制与肝细胞坏死有关。

用DSCR Cosmid DNA探针快速基因诊断先天愚型

目的 采用DSCRCosmidDNA探针作为检测先天愚型的特异性探针。方法 对 46例先天愚型患者外周血淋巴细胞分别在经过细胞培养和不经细胞培养两种处理条件下同时进行FISH检测 ,结果 在中期染色体和间期细胞核中观察到 3个杂交信号 ,杂交率均为 81%～ 95 %。结论 表明DSCRCosmidDNA探针可直接在未经培养的淋巴细胞间期核中进行FISH检测。 48h内能对先天愚型患者做出快速、准确的诊断。

用地高辛标记的染色体特异性DNA探针检测石蜡切片组织细胞中期核染色体异常的敏感方法

为了增强福尔马林固定，石蜡包埋组织切片中细胞中期核染色体原位杂交效果，我们对组织的预处理条件进行了改良，结果发现，应用微波加热，硫氰酸钠处理后蛋白酶消化时间明显缩短，减轻了对组织形态结构的破坏，在大多数切片中获得均匀一致的杂交信号。提示本改良方法可能成为肿瘤发生学研究的重要工具。

简便、敏感的地高辛标记DNA探针原位分子杂交技术

本文应用人乳头瘤病毒6B和11型基因探针原位分子杂交技术检测了57例尖锐湿疣和假性湿疣。同时用免疫组化ABC法做对照检测。探针标记选择地高辛随机引物延伸法。原位分子杂交的主要步骤包括:石蜡切片的预处理;杂交;免疫检测及显色。结果显示:在尖锐湿疣中,人乳头瘤病毒的阳性率为94.4％;免疫组化核壳抗原检测阳性率为47.2％;假性湿疣杂交结果均为阴性。结果表明:原位分子杂交方法比免疫组化方法敏感,提高了阳性率。本实验过程中遇到并试图解决的主要技术问题有:(1)防止脱片;(2)蛋白酶K的浓度;(3)显色时间的掌握;(4)左旋脒唑的剂量。

线粒体DNA与人宫颈细胞癌变的关系

目的探讨线粒体DNA与人宫颈细胞癌变的关系。方法采用PCR方法制备地高辛标记的3条人mtDNA探针,对一株人宫颈癌Hela细胞系1、例原代培养人皮肤成纤维细胞和24例子宫颈癌患者的活检癌组织及癌旁宫颈组织的染色体或间期细胞核进行了荧光原位杂交分析。结果3条mtDNA探针在原代培养人皮肤成纤维细胞染色体或间期核中均未出现阳性杂交信号;而在人宫颈癌Hela细胞和24例肿瘤组织标本的部分染色体或间期细胞核中均出现了阳性杂交信号。而在癌旁组织的部分染色体或间期细胞核中虽也出现了阳性杂交信号。但与肿瘤组织标本相比有显著性差异。结论表明癌细胞核基因组中存在mtDNA的同源序列,这些同源片段的出现可能与细胞癌变有关。

DIG标记DNA探针检测noggin在爪蟾胚胎中的表达

整体原位杂交 (Whole mountinsituhybridization)用于基因表达定位和表达分布模式的研究 ,已经成为一种非常重要的手段 .PCR方法进行探针标记 ,可以获得特异性高 ,片断大小可变的探针 .采用DIG标记 ,检测已知基因noggin在爪蟾胚胎时空分布 ,所得结果与文献报道一致 。

DHBV DNA在鸭胰脾组织中原位杂交的研究

目的：对慢性DHBV感染鸭的肝、胰、脾组织 DHBV DNA作对比研究。方法：采用原位杂交技术。结果：DHBV不仅可以感染鸭肝细胞，且可感染胰、脾组织，DHBV DNA 阳性细胞的分布密度以肝组织最高，胰脾次之。结论：DHBV具有组织器官泛嗜性，但其在肝外组织中的复制似不及在肝细胞中活跃。

Down综合征核心区Cosm id DNA探针快速基因诊断Down综合征

Objective: This study was designed to explore the diagnosis on Down syndrome by fluorescence in situ hybridization (FISH). Methods: We detected trisomy 21 with Down syndrome cristical region cosmid DNA probe, which comes from specific KU21D library of chromosome 21 (carrier is 6.7 kb, insert sequence is 35 kb) and is located in 21q22.2. 48 cases of trisomy 21 were detected on cultured and uncultured lymphoblastoid cell by FISH. Results: There were three hybridization points in interphase nuclei and three hybridization signals on metaphase three chromosome 21. Hybridization rate was 81%～95%. Conclusion: Trisomy 21 could be diagnosed rapidly and accurately by DSCR Cosmid DNA probe in uncultured lymphoblastoid interphase unclei of patients.

地高辛素标记人神经生长因子DNA探针的制备及应用

地高辛素标记人神经生长因子ＤＮＡ探针的制备及应用曹铮，陆璐，丁斐，顾静，刘飞，殷冬梅，顾晓松江苏南通医学院分子生物学研究室南通２２６００１余云开，汪家政北京军事医学科学院基础医学研究所北京１００８５０ＰＵＲＩＦＩＣＡＴＩＯＮＡＮＤＩＴＳＡＰＰＬＩＣＡ...

用生物素标记Y特异DNA探针对一例Y染色体结构异常患者的研究

用生物素标记Y特异3.4kbDNA为探针,应用染色体原位杂交技术对一例表型异常、常规染色体显带技术为小Y染色体的患者进行了研充:该患者不能与3.4kbDNA杂交,与~3H标记探针的染色体原位杂交结果一致。进一步以生物素标记此探针对患者的基因组DNA进行Southern印迹杂交分析,患者缺少一部分可检测的男性特异性片段,结合其临床特征,提示该患者为Yq的中间缺失。

生物素标记HBVDNA探针原位杂交碱性磷酸酶过氧化物酶显色检测肝内HBVDNA

本研究应用国产生物素标记HBVDNA探针,于福尔马林固定,石蜡包埋肝组织切片上,建立了碱性磷酸酶过氧化物酶显色的原位杂交技术,在此过程中发现,蛋白酶的消化是杂交能否成功的关键因素,酸、三乙醇酐、洋地黄的预处理,能促进探针参入,降低非特异背景,减低蛋白酶作用浓度或作用时间,因而提高实验稳定性、特异性。并用这一技术检测了34例肝活检和4例尸检肝组织,结果:尸肝组织均检出了HBV—DNA,其中2例核型见于肝硬化,肝癌旁组织,而2例浆型HBV—DNA见于慢活肝,亚重肝。34例肝活检组织中,只检出12例浆型HBV—DNA,其中CAH—CPH无症状携带者中检出率分别为42.9％,38.8％,33.3％,均明显高于肝硬化组织(16.7％)(P<0.05)。由此可见浆型HBV—DNA分布同病变轻重程度的关系可能并不密切,不同分布型的HBV—DNA可能只反映HBV的复制状态。