原位杂交检测微小病毒B19方法的建立及临床应用

目的 建立原位杂交（ＩＳＨ）方法检测人微小病毒Ｂ１９,并确定其在先天性心脏病９ＣＨＤ）心脏组织细胞的定位分布。方法 以Ｂ１９特异的衣壳蛋白ＶＰＩ ＤＮＡ内１１１２ｂｐ片段为模板,采用随机引物标记探针法,建立了ＩＳＨ检测Ｂ１９ ＤＮＡ浓度于０．１ｂｇ／μ１以上呈阳性。在６６例先天性心脏病心脏组织中,检测至Ｂ１９ ＤＮＡ阳性７例,并发现Ｂ１９ ＤＮＡ阳性信号主要定位于心肌细胞核内,３８例对照组健康心肌     

HBV感染与IgA肾病肾小管间质病变的关系

目的探讨ＩｇＡ肾病ＨＢＶ感染与肾小管间质病变的关系。方法利用原位分子杂交（ＨＢＶＤＮＡ）、免疫组化（ＨＢＡｇ、ＣＤ３、ＣＤ８）以及ＨＢＶＤＮＡＨＢＡｇ和ＨＢＡｇＣＤ４３双标记技术，对９１例ＩｇＡ肾病肾穿刺标本进行研究。结果肾组织内ＨＢＡｇ阳性率为６９．２％。ＨＢＶＤＮＡ原位杂交阳性率为４２９％。ＨＢＶＤＮＡ阳性的病例，双重标记染色发现ＨＢＶＤＮＡ阳性的肾小管上皮细胞可表达ＨＢｃＡｇ或／和ＨＢｓＡｇ。ＨＢＶ感染标记（ＨＢＶＤＮＡ、ＨＢｃＡｇ、ＨＢｓＡｇ）阳性组ＣＤ３阳性细胞和ＣＤ８阳性细胞数明显高于阴性组（Ｐ＜００１），并可见数量不等的Ｔ淋巴细胞入侵ＨＢｃＡｇ及ＨＢｓＡｇ阳性肾小管管壁或围绕其周围。结论感染ＨＢＶ的肾组织细胞能够表达ＨＢＡｇ，并诱导ＣＤ３阳性细胞和ＣＤ８阳性细胞浸润，从而加重肾小管、间质损害。ＨＢＶ感染对ＩｇＡ肾病的发生发展可能起着重要作用     

肝癌及癌旁组织HBVDNA原位杂交HBsAg免疫组织化学双标记研究

用原位分子杂交和免疫组化双标记技术，检测４０例肝癌（ＨＣＣ）及癌旁组织中ＨＢＶＤＮＡ和ＨＢｓＡｇ，癌及癌旁中ＨＢＶＤＮＡ阳性率为６５％，ＨＢｓＡｇ阳性率为８２．５％，表明ＨＢＶ感染与ＨＣＣ发生密切相关。二者在ＨＣＣ中表达较癌旁组织中少而弱，可能系ＨＣＣ中ＨＢＶＤＮＡ整合后复制减弱之故。发现的碎点状ＨＢｓＡｇ小包涵体可能为ＨＣＣ中ＨＢｓＡｇ的特有表现形式。小细胞ＬＣＤ中ＨＢ－ｓＡｇ表达显著高于癌旁其他类型病变，支持其更接近癌前病变的观点。     

用聚合酶链反应检测人肝细胞癌乙型肝炎病毒DNA和丙型肝炎病毒RNA

为研究乙型肝炎病毒ＤＮＡ（ＨＢＶＤＮＡ）和丙型肝炎病毒ＲＮＡ（ＨＣＶＤＮＡ）与肝细胞癌的关系，用聚合酶链反应（ＰＣＲ）和巢式ＰＣＲ（ｎｅｓｔｅｄ－ＰＣＲ）分别检测４２例肝肿瘤组织中ＨＢＶＤＮＡ和ＨＣＶＲＮＡ。结果：１例胆管细胞癌组织ＨＢＶＤＮＡ和ＨＣＶＲＮＡ均阳性，１例胆管囊腺瘤ＨＢＶＤＮＡ阳性。４０例肝细胞癌组织中，单纯ＨＢＶＤＮＡ阳性１９例，单纯ＨＣＶＲＮＡ阳性３例，二者均阳性１０例。ＨＢＶＤＮＡ阳性率７２．５％，ＨＣＶＲＮＡ阳性率３２．５％。ＨＢＶＤＮＡ和ＨＣＶＲＮＡ感染与肝癌组织学分型无关；且肝细胞癌中ＨＣＶ感染与ＨＢＶ未见相关。结果提示，我国ＨＢＶ感染仍是引起肝细胞癌的主要原因。但由于肝细胞癌患者中ＨＣＶ的感染率也较高，且有上升趋势，因此ＨＣＶ可能也是肝细胞癌发生的重要原因之一。     

肝硬变内HBV DNA及其五种抗原的表达及意义

取２２５例人肝硬变活检组织石蜡切片，检测了ＨＢＶＤＮＡ及其５种抗原。分别用免疫组化ＡＢＣ法检测ＨＢｘＡｇ、ｐｒｅ－Ｓ＿１和ｐｒｅ－Ｓ＿２抗原；用ＰＡＰ法检测ＨＢｓＡｇ和ＨＢｃＡｇ；用原位杂交方法检测ＨＢＶＤＮＡ；用免疫组化、原位杂交双标记方法检测ＨＢＶＤＮＡ和ＨＢｓＡｇ、ＨＢｘＡｇ或ＨＢｃＡｇ。结果显示，阳性检出率ＨＢｓＡｇ为７０．０％（１２８／１８３例），ｐｒｅ－Ｓ＿１抗原为６４．４％（８５／１３２例）、ｐｒｅ－Ｓ＿２抗原为６１．４％（８１／１３２例），ＨＢｘＡｇ为７５．３％（１１３／１５０例），ＨＢｃＡｇ为２２．４％（３９／１７４例），ＨＢＶＤＮＡ为６２．４％（５８／９３例）。双标阳性检出率ＨＢＶＤＮＡ和ＨＢｓＡｇ为３７．３％（１９／５１例），ＨＢＶＤＮＡ和ＨＢｘ－Ａｇ为８６．３％（４４／５１例），ＨＢＶＤＮＡ和ＨＢｃＡｇ为３９．２％（２０／５１例）。ＨＢＶＤＮＡ和ＨＢＶ５种抗原阳性病例中８０％以上均伴有肝细胞不典型增生。这一结果表明，在我国肝硬变的发生发展与ＨＢＶ慢性感染有密切的关系。     

慢性乙型肝炎肝内HBVDNA和HBsAg的双标记定位研究

应用原位杂交和免疫组化ＰＡＰ法双标记技术，结合病人乙型肝炎（乙肝）病毒血清学标志物检测结果，研究了３１例慢性乙肝病人肝穿刺组织中乙型肝炎病毒ＤＡＮ和ＨＢｓＡｇ的分布及意义。结果显示，肝辔内检ＨＶＤＮＡ２３例，ＨＢｓＡｇ２６例，二者同时检出者２１例。从同组病人肝组织的ＨＢＶＤＮＡ和ＨＢｓＡｇ双标检测结果与其乙肝病毒血清学标志物检测结果的比较来看，肝组织内ＨＢＶＤＮＡ和ＨＢｓＡｇ同时很可能表明ＨＢＶ正     

不同慢性肝病患者血清中乙型肝炎病毒DNA定量检测及其临床意义

目的探讨乙型肝炎病毒ＤＮＡ含量的临床意义。了解乙型肝炎病毒（ＨＢＶ）免疫标志不同状态的慢性肝病患者血清ＨＢＶＤＮＡ浓度及其临床意义。方法应用建立的竞争性聚合酶链反应（ＰＣＲ）方法定量检测慢性肝炎（ＣＨ）５１例、肝硬化（ＬＣ）３６例、原发性肝癌（ＰＨＣ）３８例的血清ＨＢＶＤＮＡ浓度。结果ＨＢＶＤＮＡ阳性的ＣＨ患者血清ＨＢＶＤＮＡ浓度为４．３６ｌｏｇ１０ＨＢＶＤＮＡ拷贝５０μｌ（下同），ＬＣ为４．５５，ＰＨＣ为４．４３，三组间无显著性差异（Ｐ＞０．０５）；血清ＨＢＶ五项免疫标志均阴性或抗－ＨＢｓ阳性的慢性肝病患者中，有３７．５％患者存在低水平ＨＢＶ复制；ＨＢｅＡｇ阳性患者的ＨＢＶＤＮＡ浓度总体上明显高于抗－ＨＢｅ阳性组，但其中部分患者的ＨＢＶＤＮＡ浓度也很高。结论提示ＨＢＶ的复制状态与慢性肝病的病期无明显关系；在抗－ＨＢｅ阳性的患者中存在个体差异，故不能仅依据抗－ＨＢｅ阳转来判断ＨＢＶ复制减少或停止。     

磷甲酸钠在鸭体内对鸭乙型肝炎病毒的抑制作用

以鸭乙型肝炎病毒（ＤＨＢＶ）静脉感染雏鸭为模型,分组腹腔注射磷甲酸钠（ＰＦＡ）２５０ｍｇ、１２５ｍｇ、６２．５ｍｇ／ｋｇ及生理盐水,观察治疗后鸭血清中ＤＨＢＶＤＮＡ及ＤＨＢｓＡｇ的动态变化,并检测肝、肾、脾及胰中ＤＨＢＶＤＮＡ的分布;提取肝脏中超螺旋ＤＮＡ（ＳＣＤＮＡ）,检测ＰＦＡ对ＤＨＢＶＤＮＡ复制的影响。结果表明：ＰＦＡ治疗第７天到第２１天,１２５ｍｇ和２５０ｍｇ／ｋｇ剂量组对ＤＨＢｓＡｇ有显著的抑制作用;１２５ｍｇ和２５０ｍｇ／ｋｇ剂量组治疗第１４、２１天对血清中ＤＨＢＶＤＮＡ有显著的抑制作用;１２５ｍｇ和２５０ｍｇ／ｋｇ剂量组治疗第２１天肝及肾中ＤＨＢＶＤＮＡ明显下降;２５０ｍｇ／ｋｇ剂量组治疗２１天对ＤＨＢＶ感染鸭肝细胞内ＤＨＢＶＲＣＤＮＡ、ＬＤＮＡ及ＳＣＤＮＡ的合成有明显抑制作用。可见,最大剂量２５０ｍｇ／ｋｇＰＦＡ每天２次,治疗２１天,对ＤＨＢＶ感染鸭血清中ＤＨＢｓＡｇ、血清及肝、肾中ＤＨＢＶＤＮＡ都有抑制作用,对脾、胰中ＤＨＢＶＤＮＡ抑制不明显。提示该药能抑制ＤＨＢＶ感染,但未能清除病毒,故停药后可出现反跳现象。     

HBV感染与IgA肾病肾小管-间质病变的关系

目的 探讨ＩｇＡ肾病ＨＢＶ感染与肾小管间质病变的关系。方法 利用原位分子杂交（ＧＨＢＶ ＤＮＡ）、免疫组化（ＨＢＡｇ、ＣＤ３、ＣＤ８）以及ＨＢＶ ＤＮＡ－ＨＢＡｇ和ＨＢＡｇ－ＣＤ４３双标记技术,对９１例ＩｇＡ肾病肾穿刺标本进行研究。结果 肾组织内ＨＢＡｇ阳性率为６９．２％。ＨＢＶ ＤＮＡ原位杂交阳性率为４２．９％。ＨＢＶ ＤＮＡ阳性的病例,双重标记染色发现ＨＢＶ ＤＮＡ阳性肾小管上皮细胞可表达ＨＢ     

免疫刺激DNA及IL-2表达质粒对乙肝病毒DNA疫苗诱导抗体的影响

分别将含免疫刺激ＤＮＡ序列（ＩＳＳ）的质粒ｐＵＣ１９、ｐｃＤＮＡ３及编码ＩＬ ２的真核表达质粒ｐｃＤＮＡ３ ＩＬ ２,与乙肝病毒ＤＮＡ疫苗共同注射于小鼠胫前肌内。定量检测免疫小鼠体内ＨＢｓＡｇ的表达及血清中抗ＨＢｓ的水平。结果显示：ｐＵＣ１９、ｐｃＤＮＡ３及ｐｃＤＮＡ３ ＩＬ ２对乙肝病毒ＤＮＡ疫苗在肌肉内表达ＨＢｓＡｇ无影响或有抑制,但它们均能显著促进疫苗诱导抗体的产生（Ｐ＜００１）,抗体水平的增加与ＩＳＳ的数量呈剂量反应关系。     

Prevalence of human parvovirus B19 DNA in cardiac tissues of patients with congenital heart diseases indicated by nested PCR and in situ hybridization.

BACKGROUND: Infection with parvovirus B19 (B19) was reported to be correlated with myocarditis, cardiomyopathy, and kawasaki disease. But no information is available about the relationship between inutero B19 infection and congenital heart disease (CHD). OBJECTIVE: To explore whether there is relationship between B19 infection and CHD. STUDY DESIGN: Retrospective investigation of biopsy samples from CHD patients from January 1996 to December 1998. METHODS: Parvovirus B19 was detected in biopsy samples from 42 cases of CHD patients and 38 cases of biopsy or autopsy samples from patients with other diseases (controls) by nested polymerase chain reaction (PCR) and in situ hybridization (ISH) technique. HE staining was also performed to observe the morphology of these cardiac tissue samples. RESULTS: Nested PCR assay indicated that seven of 42 (16.7%) CHD cardiac tissue were B19 DNA positive, while all the 38 controls were B19 DNA negative, the difference is significant (P = 0.012). ISH assay indicated that five of 42 (11.7%) CHD cardiac tissues were positive for B19 DNA and none of the control cardiac tissue were positive, all B19 DNA positive signals were located in the nucleuses of cardiac cells. HE staining showed that there was no inflammatory change in B19 DNA positive cardiac tissue. CONCLUSIONS: Parvovirus B19 DNA was presented in part of CHD cardiac tissues and located in nucleus, which suggested that inutero B19 infection might be correlated with CHD.     

先天性心脏病患者B19等病原感染的调查研究

Objective　To explore relationship of Parvovirus B19(B19), Toxoplasma gondii(TOX), rubella virus(RV), cytomegalovirus (CMV) or herpes simplex virus type-2 (HSV-2) infection with congenital heart disease (CHD). Methods　We conducted a case-control study on detection of B19, TOX, RV, CMV or HSV-2 gene in cardiac tissue of 66 cases of CHD and 38 cases controls with polymerase chain reaction (PCR). Results　1) The positive rates of B19, TOX, CMV and HSV-2 in 66 cases CHD were 18.2%, 15.2%, 25.8% and 4.5%, respectively, while that in 38 cases control groups were 0%, 2.6%, 21.2% and 2.6%, reapectively. The positively of B19, TOX was significantly different (P<0.05) between CHD and control groups, while no statistical difference (P<0.05) for CMV and HSV-2. 2) Seven of 30 (23.3%) CHD were positive for RV-RNA, compared with control group which was all negative (P=0.0327). Conclusion　The results show that B19, TOX, RV infection mighet be important risk factors for CHD, while CMV and HSV-2 had no relationship with CHD.     

Detection of parvovirus B19 in macerated fetal tissue using in situ hybridisation.

AIMS: To compare the application of a non-radioactive in situ hybridisation (ISH) technique with an immunocytochemical technique for the detection of human parvovirus B19 in formalin fixed, paraffin wax embedded sections of macerated fetal tissue. METHODS: Archived samples of liver, lung or kidney from 19 human fetuses were investigated for parvovirus B19 using a full length digoxigenin labelled DNA probe of 5.5 kb; bound probe was detected using an anti-digoxigenin (alkaline phosphatase) conjugate and visualised using NBT/BCIP. Immunocytochemical detection of parvovirus B19 was performed using a monoclonal mouse antiparvovirus B19 antiserum, with a streptavidin-biotin complex (horse radish peroxidase) method. Cases were selected to provide a range of diagnostic certainty and a range of degrees of macerative degeneration. RESULTS: Parvovirus B19 was found in 15 of 19 cases using the B19 ISH technique compared with 8 of 19 cases using the immunocytochemical technique. The four negative cases were all controls known to be parvovirus B19 free. All ISH positive cases showed excellent staining with low background regardless of extent of maceration and tissue type. In comparison, sections stained by the immunocytochemical method showed considerable non-specific immunoreactivity in many cases, particularly with severe maceration. Kidney and lung tissues gave the cleanest results. CONCLUSIONS: ISH is more effective than the immunocytochemical technique for the detection of human parvovirus B19 in macerated fetal tissue. The lack of detectable background staining with the ISH technique led to easier interpretation suggesting that this technique should be the method of choice for the investigation of parvovirus B19 in macerated postmortem tissues.     

PCR-based diagnosis of enterovirus and parvovirus B19 in paraffin-embedded heart tissue of children with suspected sudden infant death syndrome

The diagnosis of viral myocarditis remains difficult and generally depends on clinical and histologic criteria. Viral cultures and serology are often unrewarding with low yields. The purpose of this study was to analyze the usefulness of PCR in the rapid diagnosis of myocarditis in children. PCR was used to analyze 120 myocardial tissue samples from 60 cases of sudden infant death syndrome (SIDS) and 56 myocardial tissue samples from 36 cases with well-known causes of sudden death (11 children younger than 1 year and 25 children 1-10 years old). The myocardial tissue samples were evaluated for the presence of enteroviruses and parvovirus B19 using PCR primers designed to consensus and unique sequences of these viral genomes. Enteroviruses could be detected in 14 cases of SIDS, whereas the detection of enteroviral nucleic acid within the control group was negative. Seven cases with myocardial infection caused by parvovirus B19 were found in the SIDS group. The detection of parvoviruses in the control group of the 11 children younger than 1 year was negative, whereas 3 positive cases of parvoviruses could be detected in the control group of children from 1 to 10 years old. In the myocardial sample of one SIDS case, both enteroviruses and parvovirus B19 could be detected. Our results emphasize the importance of modern molecular biologic methods in cases of sudden infant death even when conventional histologic examination revealed no serious findings in heart muscle tissue.     

Parvovirus B19 infection in thoracic organ transplant recipients.

BACKGROUND: Clinical manifestations of parvovirus B19 infection in immunocompromised patients are mostly reported as acute or chronic hematologic disorders. More recently, respiratory or renal involvement has been described. OBJECTIVE: We started in 1994 a prospective study of parvovirus B19 infection in a group of lung (LTP) and heart-lung (HLTP) transplanted patients, including occasionally heart transplanted (HTP) patients. STUDY DESIGN: 62 patients (49 LTP, 11 HLTP, 2 HTP) were included in a serological survey and DNA detection by PCR was performed on each serum sample of the first 29 patients; later we performed it only when serology could suggest an acute episode, or when parvovirus infection could be suspected on clinical or biological observations. A total of 1655 sera were examined by serological tests and DNA detection was done in 500 samples. Specific IgM, seroconversion, significant increase of specific IgG levels, and/or parvovirus B19 DNA detection, were considered as markers of viral infection. RESULTS: We observed the presence of both markers of infection in 24 patients (39%), with an individual combination of positive antibody and PCR results. Acute or chronic anaemia, neutropenia were associated to these laboratory findings in 19 patients, but in five cases, an asymptomatic clinical infection suggested viral persistence. CONCLUSIONS: We report parvovirus associated acute or chronic anaemia and pancytopenia in a group of LTP, HLTP and HTP patients, as well as asymptomatic cases of infection. In the hypothesis of a parvoviral persistent or latent infection, current diagnosis methods may be unreliable to identify any other clinical manifestations.     

先天性心脏病心脏组织中TORCH病原基因的检测

目的 探讨先天性心脏病（CHD）心肌组织中TORCH各病原体（弓形体／TOX;风疹病毒／RV;单纯疱疹病毒／HSV2;巨细胞病毒／CMV）的感染状况。方法 通过收集42例先天性心脏病及38例对照组的心脏组织石蜡标本,用聚合酶链反应（PCR）或逆转录－聚合酶链反应（RT－PCR）技术进行病毒基因的检测。结果 在42例先天性心脏病心肌组织中检测到CMV、HSV、TOX的阳性率分别为26.2%、4.7%和16.7％,而例对照组中CMV、HSV2、TOX的阳性率分别为21.1%、2.6%和2.6%,丙组比较TOX在两组中的检测差异有显著性（P＝0.0378）,而CMV、HSV2在两组中无统计学意义（P＞0.05）。在30例先天性心脏病中RV的阳性率为23.3%,而20例对照均有性,两组间差异有显著性（P＝0.0328）。结论 从先天性心脏病的心脏组织中发现有TORCH病原体TOX、RV、CMV和HSV2基因存在,为从分子水平进一步进行TORCH病原感染与先天性心脏病关系的研究打下基础。     

Persistence of parvovirus B19 DNA in synovium of patients with haemophilic arthritis

A progressive arthropathy develops commonly in haemophiliacs and its pathogenesis is not fully understood. Human parvovirus B19 has been associated with several diseases including acute and chronic arthropathy and some studies suggest its implication in chronic inflammatory diseases of the joints such as rheumatoid arthritis. In haemophiliacs parvovirus B19 infection occurs very frequently because of its transmission with plasma derivatives. In order to assess a role of B19 virus in haemophilic arthritis, synovial tissue samples from patients with haemophilia with arthritis and from patients, nonhaemophiliacs, with arthrosis or with joint trauma were examined for B19 DNA by nested PCR. In addition, the prevalence of antibody to parvovirus B19 NS1 protein as a possible serological marker of persistent B19 infection was tested and the association of the outcome of parvovirus infection with genetic diversity of B19 P6 promoter sequences was investigated. B19 DNA was detected in the synovial tissue of 31% of haemophiliacs with progressive arthropathy and of 5% of control patients. Fourteen out of 17 patients (82%) with haemophilic arthritis and with B19 DNA in their synovial membranes had IgG antibodies against the nonstructural protein NS1 of parvovirus B19. On the other hand, 19% of patients with haemophilia with B19 PCR negative synovial tissue and 21% of controls showed anti-NS1 antibodies. The P6 promoter presented specific sites of point mutations shared frequently by isolates from patients with haemophilia and arthritis. These results indicate that B19 DNA can persist in the synovial membranes of patients with haemophilic arthritis significantly more frequently in comparison to control individuals with arthrosis or joint trauma and show a correlation between anti- NS1 antibody presence and B19 DNA persistence in the synovial tissue.     

Fetal erythropoiesis and parvovirus B19]

Target cells for the human parvovirus B19 include erythroid progenitors located in the bone marrow in adults and in the liver in fetuses of 12 to 30 weeks gestational age. The main manifestations of fetal parvovirus B19 infection seem to be related to lysis of the erythroid progenitors which causes non immune hydrops fetalis with severe anemia, congestive heart failure, generalized edema and death. The exact incidence of human parvovirus B19 infection during pregnancy remains unclear.     

Cytokine gene polymorphisms associated with symptomatic parvovirus B19 infection

BACKGROUND: The immune system has been implicated in the pathogenesis of certain clinical manifestations of parvovirus B19 infection, including rash and arthralgia. Cytokines feature in the pathogenesis of parvovirus B19 infection, so inherited variability in cytokine responses to B19 infection might have a bearing on the symptomatology of parvovirus B19 infection. AIMS: To investigate the possible role of cytokine gene polymorphisms in the clinical manifestations of parvovirus B19 infection. METHODS: Thirty six patients with a variety of symptoms at acute infection and follow up (mean, 22.0 months) and controls (99-330, depending on the locus) were examined for the following cytokine polymorphisms: tumour necrosis factor alpha (TNF alpha), -308; interferon gamma (IFN-gamma), +874; interleukin 6 (IL-6), -174; IL-10, -592, -819, and -1082; and transforming growth factor beta1 (TGF beta 1), +869 (codon 10) and +915 (codon 25). RESULTS: The TNF alpha -308*A allele occurred in 13.9% of the parvovirus group compared with 27.0% of the control group (odds ratio (OR), 0.44; p = 0.02). The TGF beta 1 CG/CG haplotype was more frequent in the parvovirus group than in the controls (16.7% v 5%; OR, 4.8; p = 0.02). Within the B19 infected group, the TGF beta 1 +869 T allele was associated with skin rash at acute infection (p = 0.005), whereas at follow up the IFN-gamma +874 T allele was associated with the development of anti-B19 non-structural protein 1 antibodies (p = 0.04). CONCLUSIONS: The results of the present study suggest that inherited variability in cytokine responses may affect the likelihood of developing symptoms during parvovirus infection.