血清HBV标志物阴性肾炎肾组织中HBV-DNA的原位杂交检测

目的 对血清HBV标志物阴性肾炎患者肾组织进行HBV—DNA的检测，为临床诊疗提供理论依据。方法 应用原位杂交（ISH）技术检测37份血清HBVAg阴性和18份血清HBVAg阳性的肾炎患者肾活检石蜡包埋组织切片中的HBV—DNA。结果 在血清HBVAg阳性的18例标本中HBV-DNA的检出率为88．9％（16／18），血清HBVAg阴性37例中检测出HBV—DNA5例（13．5％）。原位杂交显示两组检出的HBV-DNA均以肾小管上皮细胞质分布为主，与组织中HBVAg阳性颗粒的分布基本一致。结论 血清HBVAg阴性的HBV相关性肾炎临床上并非少见，应给予足够的关注。HBV—DNA在肾组织中的检出，表明HBV在HBV相关性肾炎的发病机理中有着重要作用。     

原位杂交法检测肝组织中丁型和乙型肝炎病毒核酸

利用国外引进的重组质粒获得纯化基因片段，分别以随机引物法和ＰＣＲ法制备地高辛素标记的ＨＢＶＤＮＡ探针和ＨＤＶｃＤＮＡ探针。用原位杂交法检测了石蜡包埋的肝组织切片ＢＶＤＮＡ和ＨＤＶＲＮＡ。４９例感染肝组织分为两组：丁肝组２３例；单纯乙肝组２６例，ＨＢＶＤＮＡ的检出率丁肝组（７８．２６％）与乙肝组（７６．９２％）无统计学差异；而ＨＤＶＮＡ的检出率丁肝组（６０．８７％）明显高于乙肝组（１５．３８％）。ＨＢＶＤＮＡ可见于受染肝细胞的胞核或胞浆内，而ＨＤＶＲＮＡ绝大部分见于肝细胞胞核。两种病毒核酸阳性细胞在肝组织中的分布特点大致相同：弥漫或散在地分布于肝小叶或假小叶内，或局灶性分布于小叶周边。ＨＤＶＲＮＡ阳性的肝组织都或多或少地同时存在ＨＢＶＤＮＡ。同一例肝组织中，ＨＢＶＤＮＡ阳性细胞从数量和颗粒密度上似略高于ＨＤＶＲＮＡ。将乙肝组和丁肝组两组病人肝内ＨＢ－ｓＡｇ、ＨＢｃＡｇ和ＨＢＶＤＮＡ及血清ＨＢｅＡｇ作了比较，各指标阳性率虽有差异，但均无统计学意义。因此，未发现ＨＤＶ感染对ＨＢＶ的复制有明显抑制作用。此结果对以往用血清学或免疫组化方法对ＨＤＶ的研究有所补充和深入，亦可为研究其它类型病毒性肝炎之间的重叠感染所借鉴。     

Detection of the Epstein-Barr virus in primary gastric lymphoma by in situ hybridization

The Epstein-Barr virus (EBV) has been shown to be associated with numerous human malignancies including Burktt's lymphoma and nasopharyngeal lymphoepithelioma. In addition, some typical gastric adenocarcinomas were also recently reported to demonstrate EBV relevance. The present study was designed to detect EBV in primary gastric lymphoma, using the in situ hybridization (ISH) method, in which oligo-nucleotide probes for the EBERl RNA and the EBV DNA W region have been used. Of the 49 cases of primary gastric lymphoma studied, which all showed B cell immunopheno-type, EBER1 sequences could only be found in four cases, including two low-grade cases and two high-grade cases of histological subtypes while the number of positive cells was less than 50% of the tumor cells. In one case of low-grade mucosa associated lymphoid tissue (MALT) lymphoma, the EBER1 -positive neoplastic cells were found in the regional lymph node, but the primary site of the stomach showed no positive signals. The EBV presence was further confirmed by the EBV DNA ISH. Using the ISH method, rare or occasional positive lymphoid cells (probably non-tumorous bystander cells) could be detected in 10 other cases including all histological subtypes. The present study shows that only a small proportion of primary gastric lymphoma is associated with EBV, and such positive cases could be found in both high- and low-grade histological subtypes. It is also suggested that the EBV presence in the neoplastic cells of some cases of primary gastric lymphoma is most likely a secondary phenomenon.     

Detection of hepatitis B virus genome in hepatocellular carcinoma tissues with PCR-in situ hybridization.

The detection is described of hepatitis B virus (HBV)-DNA in preserved hepatocellular carcinoma tissues, which were derived from 14 HBV-seropositive patients. Detection was by polymerase chain reaction (PCR) amplification of the target sequence, followed by specific localization of the PCR product with in situ hybridization. PISH (PCR-in situ hybridization) yielded strong positive signals in most of the tumor tissues despite very low copy numbers of chromosome-integrated HBV genome, whereas no signal was detected in control samples, indicating that the signals were specific for HBV. Positive signals were sometimes detected in cirrhotic nodules surrounding the tumor regions, indicating that HBV had infected non-transformed liver cells. HBV-DNA was detected in both nucleus and cytoplasm in some specimens, possibly representing HBV at different stages of the life cycle. In one case, a gradient of viral DNA was revealed, with the highest DNA signal centered at the site of viral antigen expression. Taken together, PISH is shown to be a highly sensitive molecular detection method that is capable of detecting the presence of a low copy number viral genome in situ.     

丁型病毒性肝炎的原位分子杂交及免疫组化研究

利用原位分子杂交和免疫组化方法对１４２例乙型肝炎活检肝组织进行丁型肝炎病毒ＲＮＡ及其抗原的定位研究。２８／１４２例丁型肝炎病毒标记阳性。其中慢性重症型６例；慢性活动性１７例；慢性持续性５例。慢性活动性乙肝重叠丁型肝炎病毒感染组发生早期肝硬变的比例明显高于无重叠感染组（Ｐ＜０．０５）。２８例丁型肝炎病毒感染肝组织１９例ＨＢｃＡｇ阳性，并以核浆型为主，提示活动性ＨＢＶ复制与ＨＤＶ感染的正相关性，两者相加作用导致肝损害加重并加速发展为肝纤维化。ＨＤＶＲＮＡ在肝细胞内大量蓄积，ＨＤＡｇ在碎屑状坏死边缘肝细胞或气球样变肝细胞内呈浆膜型分布，提示ＨＤＶ直接细胞毒在丁型肝炎发病学中的作用。     

Detection of hepatitis B virus genome in hepatocellular carcinoma tissues with PCR-in situ hybridization

The detection is described of hepatitis B virus (HBV)-DNA in preserved hepatocellular carcinoma tissues, which were derived from 14 HBV-seropositive patients. Detection was by polymerase chain reaction (PCR) amplification of the target sequence, followed by specific localization of the PCR product with in situ hybridization. PISH (PCR-in situ hybridization) yielded strong positive signals in most of the tumor tissues despite very low copy numbers of chromosome-integrated HBV genome, whereas no signal was detected in control samples, indicating that the signals were specific for HBV. Positive signals were sometimes detected in cirrhotic nodules surrounding the tumor regions, indicating that HBV had infected non-transformed liver cells. HBV-DNA was detected in both nucleus and cytoplasm in some specimens, possibly representing HBV at different stages of the life cycle. In one case, a gradient of viral DNA was revealed, with the highest DNA signal centered at the site of viral antigen expression. Taken together, PISH is shown to be a highly sensitive molecular detection method that is capable of detecting the presence of a low copy number viral genome in situ.     

生物素标记探针原位杂交法检测细胞中c-myc基因的表达

本文采用生物素标记的c-mye基因作探针，通过细胞原位分子杂交技术，对人舌癌、膀胱癌和正常细胞中c-myc基因的转录量进行了定性和半定量分析。结果表明与正常细胞相比，舌癌和膀胱癌细胞中c-myc基因异常高度表达。     

肺癌中EB病毒的原位杂交检测

为观察EB病毒在肺癌组织中的分布并探讨EB病毒感染与肺癌的关系，对87例肺癌组织中经多聚酶链反应（PCR）检测52例EB病毒阳性的石蜡包埋组织，用原位杂交技术进行定位检测。癌组织及癌旁肺组织原位杂交EB病毒阳性率分别为37．9％（33／87）和11．5％（IO／87）；中、低分化癌与高分化癌EB病毒阳性率有显著差异，强阳性病例均为分化较差的肿瘤；累及胸膜、肋骨、隔肌的肺癌，其EB病毒阳性率明显高于未侵及者；随着EB病毒感染的加重，淋巴细胞浸润增多。结果表明：EB病毒感染与肺癌细胞的生物学特性及肺癌的发展有关。     

原位杂交法检测非甲～庚型肝炎患者肝组织中TT病毒DNA

目的 证实在非甲－庚型病毒性肝炎患者肝组织中TT病毒（transfusion-transmitted virus,TTV)的存在。方法 采用地高辛素标记TTV DNA探针以原位杂交技术对51例血清学病毒标记非甲－戊型、免疫组化检测肝组织中HBsAg、HCV NS3Ag及HGV N55Aag阴性的病毒性肝炎患者石蜡包埋肝组织进行了检测。结果 各型病毒性肝炎肝组织中TTV基因的总检出率为27．5％,其中急性轻型肝炎的检出率为30．8％（4 ／13）,急性重型肝炎为1／8,亚急性重型肝炎为3／7,慢性肝炎为2／6,活动性肝硬化为2／9,慢性重肝肝炎为1／4,原发性肝癌为1／4。TTV DNA表达于肝细胞核或胞质内,以核型多见。在急性肝炎,TTV阳性细胞弥漫分布于肝小叶内,慢性肝炎于汇管区附近较为密集,而在肝硬化病例,阳性细胞在假小叶内多呈片族状不规则分布。结论 在不明原因病毒性肝炎患者血清及肝组织中TTV DNA的检出表明TTV为一种新型的肝炎病毒,TTV为一种嗜肝性病毒。     

Efficiency of in situ hybridization as a function of probe size and fixation technique

In an attempt to improve fixation technique for viral RNA detection by in situ hybridization, we have quantitatively compared the hybridization signal obtained when measles virus or visna virus infected cell cultures were fixed with eight different fixatives and hybridized with 35S-labeled virus-complementary DNA probes of several size ranges. Small probes (mean length, 70 bases) gave higher signals than larger probes (mean lengths 140, 350, and 780 bases) with all fixatives. This increase in signal was minimal with acetic ethanol or formalin, but was dramatic with fixatives containing glutaraldehyde; with these fixatives the signals with small probes were 6.5- to 22-fold greater than with large probes. The highest signals were obtained with periodate-lysine-paraformaldehyde-glutaraldehyde (PLPG) fixed cells hybridized with small probes, and were 1.5- to 6.7-fold greater than those obtained with the commonly used fixative acetic ethanol. PLPG and other glutaraldehyde based fixatives also greatly improved the preservation of cellular morphology compared to acetic ethanol.     

Detection of Epstein-Barr virus in lymphoid tissues of patients with infectious mononucleosis by in situ hybridization.

Although the immunological response during infectious mononucleosis (IMN) has been studied in detail, little is known about the spread of Epstein-Barr virus (EBV) in lymphoid organs or the topographical distribution of the infected cells. In this study, EBV was detected in 11 lymph nodes, 4 tonsils, and 1 spleen of 16 patients with IMN. The predominant cell type positive for the EBV genome was identified as small lymphocytes localized chiefly within typical T areas, preferentially in perifollicular and interfollicular regions of the lymph node. A few endothelia of epithelioid venules were also found to be positive. Furthermore, a small number of sinus lining cells of lymph nodes exhibited labelling. Altogether, only a small number of cells, not exceeding 1 per cent of all cells, were infected with EBV. Our results show that only a small number of lymphocytes carry the EBV and that besides B lymphocytes, other cell constituents of lymphatic tissues are infected by EBV during IMN.     

Detection and localization of human papillomavirus DNA in human genital condylomas by in situ hybridization with biotinylated probes

We have examined the distribution of human papillomavirus (HPV) DNA in paraffin sections of humans warts by in situ hybridization with biotin-labeled DNA probes. Recombinant plasmid DNAs (HPV-1, -6, -11, -16) were labeled by nick translation with biotinylated deoxyuridine triphosphate. Paraffin sections were hybridized with the probes for 18 h in stringent or non-stringent conditions, and DNA-DNA hybrids were detected by immunocytochemistry. Paraffin sections of warts were also examined for the presence of HPV capsid antigen with the avidin-biotin peroxidase complex method for immunocytochemistry. HPV DNA was detected and localized in paraffin sections from a plantar wart, a laryngeal papilloma, and seven anogenital condylomas. The specific HPV type present in each lesion was determined by hybridization under stringent conditions with the homologous DNA probe. The papillomas were found to contain many more cells with replicating virus DNA, as demonstrated by in situ hybridization, than was apparent from the number of cells containing detectable virus antigen. In situ hybridization with biotin-labeled probes is an effective technique for the identification of HPV infection in routinely collected and processed tissue specimens.     

Detection of herpes simplex virus DNA by in situ hybridization technique and polymerase chain reaction in Papanicolaou-stained cervicovaginal smears

Papanicolaou-stained cervicovaginal smears from six patients with herpes simplex virus (HSV) infection were destained and reprocessed by means of in situ hybridization (ISH) technique to demonstrate the presence of HSV DNA utilizing biotinylated probe. Positive results were obtained in all six cases with intense staining signal for the HSV DNA in the nuclei of cells having a ground-glass nuclear appearance as well as in multinucleated giant cells. Furthermore, a hybridization signal was also noted in smears that had been prepared as much as 3 yr previously. HSV type 2-specific antigen was confirmed in six destained smears by means of immunoperoxidase staining. Moreover, polymerase chain reaction (PCR) was also performed for four patients from Pap-destained cervicovaginal smears. Amplified HSV DNA was detected in all four cases as 106 basepair PCR products by polyacrylamide gel electrophoresis. The combined use of cytology and the ISH technique and PCR amplification was of great value for the rapid cytodiagnosis of genital infection of HSV.     

52 例病毒性肝炎患者肝组织中丙型肝炎病毒 RNA 和 HCAg-NS3 的测定

应用地高辛标记探针原位杂交法和单克隆抗ＨＣＶ－ＮＳ３－ＨＲＰ建立直接酶标免疫组化法分别测定５２例肝炎患者肝组织ＨＣＶＲＮＡ和ＨＣＡｇ－ＮＳ３。结果抗ＨＣＶ阳性组ＨＣＶＲＮＡ检出率５７．１％（１６／２８），ＨＣＡｇ－ＮＳ３检出率５３．６％（１５／２８）；抗ＨＣＶ阴性组其两项检出率均为１２．５％（３／２４）。肝组织中ＨＣＶＲＮＡ阳性物呈蓝紫色细小颗粒存在于肝细胞核或胞浆内，其在肝小叶中的分布可分为３型，即弥漫型、局灶型、散在型。肝组织中ＨＣＡｇ－ＮＳ３阳性物呈棕黄色细小颗粒分布于肝细胞核或胞浆内，以单个或数个阳性细胞散布于肝小叶中。２３例ＨＣＶＲＮＡ或／和ＨＣＡｇ－ＮＳ３阳性病例以肝炎后肝硬化（ＬＣ）病例占多数（１４／２３），其次为慢性重型肝炎（ＣＳＨ）和中度慢性肝炎（ＣＡＨ）。此两种检测方法具有较高符合率（９０．４％，４７／５２），表明病毒核酸及其表达产物均存在于肝细胞内，与ＨＣＶ感染密切相关。这为ＨＣＶ感染诊断提供了直接依据，有利于研究ＨＣＶ感染中病毒复制、慢性化进程、抗病毒治疗监测及重叠感染时病毒相互关系。     

A case of 46,XX,r(X) (p1q1) diagnosed by in situ hybridization

A small marker chromosome was identified as an X-derived ring chromosome by in situ hybridization with a biotinylated X-chromosome specific a-satellite DNA probe. This procedure clearly determined the chromosomal origin of the marker chromosome, which had been impossible to define by conventional cytogenetic techniques including high resolution banding.     

Detection and localization of an extra HLA locus in a karyotypically normal male by chromosomal in situ hybridization

The codominant expression of three HLA haplotypes was found in a healthy 21-year-old Black male, whose prometaphase karyotype was normal by light microscopy. He was the sibling of an antenatally diagnosed female fetus with a partial duplication of 6p. The duplication arose from a complex presumably balanced maternal chromosome rearrangement: 46,XX,dir ins(14;6)(14pter----14p11::6p22----6p21.1::14 p11----14qter; 6pter----6p22::6p21.1----6qter). Chromosomal in situ hybridization using a tritium-labeled genomic clone corresponding to a class I HLA gene revealed two sites of hybridization: at 6p21.3, the band to which this probe has been assigned in normal individuals (Morton et al. 1984a) and a second site at 6p11. We postulate that a recombinational event during meiotic pairing in the mother led to the reintroduction into the normal chromosome 6 homolog of a small segment of the original insertion in chromosome 14 which contained the HLA-A and -B determinants.     

Non-isotopic in situ hybridization at the ultrastructural level

Non-isotopic in situ hybridization techniques are becoming increasingly widely used at the ultrastructural level, permitting rapid localization of nucleic acid targets with a high degree of resolution. Technical considerations dictate that the great specificity of the method cannot be matched by a similar degree of sensitivity; the value of non-isotopic ultrastructural in situ hybridization lies in its unique ability to localize nucleic acid targets in relation to submicroscopic cellular structures. This article presents an overview of non-isotopic ultrastructural hybridization methods and applications.     

DNA: DNA hybridization method for the diagnosis of hepatitis B infection

Hepatitis B viral (HBV) DNA was detected in a hepatoma cell line which produces hepatitis B surface antigen (HBsAg) and in patients with acute hepatitis B. The serum of one patient with acute hepatitis B was found to be infectious when injected i.v. into a chimpanzee up to a dilution of 10(-8). Hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) were detectable in the same serum sample by radioimmunoassay up to a dilution of 10(-5) and of 10(-3), respectively. Using DNA: DNA hybridization on nitrocellulose membranes, HBV DNA sequences were detectable up to 10(-8) dilution corresponding to the infectivity level. Based on this finding, it appears that DNA: DNA hybridization is the most sensitive method for detecting hepatitis B virus (HBV) infection. In situations with low virus levels it may be the only indicator of the presence of infectious hepatitis B virus. The use of a tritium-labelled probe makes the method economical and adaptable to hospital laboratories.     

Detection of Herpes simplex virus DNA in spontaneous abortions from HIV-positive women using non-isotopic in situ hybridization

The purpose of this study was to determine the prevalence of Herpes simplex virus (HSV) endometritis in spontaneous abortions in HIV-positive women using non-isotopic in situ hybridization (NISH). Post-abortal endometrial curettings from 18 HIV-positive women were investigated for the presence of HSV-1 and HSV-2 DNA with NISH. In addition, 18 unselected post-abortal endometrial curettings in HIV-negative women were used as controls, together with samples of normal proliferative and secretory endometrium. Thirteen of the 18 specimens (72 per cent) from the HIV-positive study group demonstrated the presence of HSV DNA, while 2 of the 18 HIV-negative group (11 per cent) showed a positive signal. Although the prevalence of HSV endometritis in the HIV-positive group was significantly higher than in the HIV-negative group (P<0.05), a causal role for the virus in inducing the abortion remains to be determined. In addition, the significance of HSV endometritis with regard to the clinical management of HIV-positive patients is as yet uncertain.     

肺癌组织中EB病毒感染的检测

目的探讨原发性肺癌中EB病毒（Epstein—Barr virus，EBV）的存在情况及EBV与原发性肺癌的关系。方法唐山市人民医院和开滦医院病理科储存的2001--2006年肺癌手术切除石蜡包埋肺癌组织108份，癌旁组织22份，以EBV阳性鼻咽癌组织为阳性对照，用原位杂交法（ISH）检测肺癌患者石蜡包埋组织标本中EBV编码的小RNA（EBERl），并采用图像分析法进行形态学定量。结果癌组织及癌旁组织EBERl的阳性率分别为33．3％（36／108）和4．5％（1／22），二者间差异有统计学意义（P〈0．01）。鳞癌、腺癌、小细胞癌及大细胞癌中EBV感染率分别是35．9％（14／39）、31．6％（12／38）、31．0％（9／29）和1／2。EBV感染与患者年龄、性别和组织学类型无关，但与肺癌的部位、癌组织分化程度有关，右肺明显高于左肺，中低分化癌明显高于高中分化癌。结论唐山地区原发性肺癌组织中EBV感染率为33．3％，EBV感染可能是肺癌的潜在病因之一，在癌组织分化的不同阶段有不同的作用。