关键词 柯萨奇病毒 克山病 原位核酸杂交
Morphological feature of positive hybridization signal
of Coxsackievirus RNA in the myocardium specimens of
Keshan disease patients and its relationship with the occurrence of the disease
Ren Liqun，Li Guangsheng，Li Xiangjun，et al.
Dep.of Pathology，Institute of Biological
【Abstract】 Objective To investigate the morphological feature of positive hybridization signal of Coxsackˉievirus RNA in the myocardium specimens of Keshan disease（KD）and the relationship between the viral infection and the occurrence of the disease.Methods Using in situ hybridization to detect Coxsackievirus RNA and its distriˉbution in65specimens of KD patients（12acute，33subacute and20chronic cases）.Results The positive hybrid signals were obtained in83%，87.9%and80%of tissue samples of acute，subacute and chronic KD，respectively.Granules of positive hybrid signal were thick，and had clear border in the myocardium of acute and subacute KD，and showed relatively localized distribution.In chronic case，most granules of positive hybrid signal were found to be disˉtributed in all part of the myocardium;They were small and with unclear border.Conclusion It is found feasible to use on situ hybridization method to perform retrospective studies on paraffin-embedded myocardial tissue even after a long-term preservation.There is a strong link between most of the specimens of KD patients and the infection with Coxsackievirus，so viral infection is an important component of multiple etiology of KD.Key words Coxsackievirus Keshan disease nucleic acid hybridization in situ Keshan disease（KD）is an endemic cardiomyopaˉthy that affects thousands ID infants，children and womˉen of child-bearing age residing in the selenium-deˉficient regions of China.The basic pathological characˉteristic of KD is multifocal necrosis（Fig.1）and reˉplacement fibrosis of myocardium，whichthen results in acute or chronic heart failure ［1］ .According to the mode ofonset and the state of cardiac function，four types of KD can be identified clinically:a）acute（sudden onset with cardiogenic shock and severe arrhythmia）;b）subaˉcute（with both cardiogenic shock and congestive heart failure）;c）chronic（with marked dilation of the cardiac chamber and congestive heart failure）;d）latent（with various degrees of cardiomegaly，accompanied by comˉpensated cardiac function）.Progression from the latent type to overt KD may or may not occur.To date，one of the most popular etiologic theories postulates multiple causality，i.e.，a possible association between enterovirus infection and deficiencies of some micronutrients，such as selenium and vitamin E.
In regard to the detection of enteroviral RNA in the myocardium samples of KD patients，Zhong et al ［2～6］ reˉported positive signals for CoxsackievirisB3in61.5%to90%of their work was based on investigations of auˉtopsy specimens of KD patients collected from endemic areas of Helongjiang（acute and chronic） ［4，5］ ，Yunnan（subacute） ［2］ ，Shandong and Yunnan（chronic）reˉgions ［3，6］ .The authors did not pay much attention to the cases occurring in the Jilin Province where there were aˉcute，severe KD outbreaks towards the end of1950s and especially in the early days of1960s.There is no inforˉmation as to the presence or absence of morphologic feaˉtures of positive signals of enterovirus RNA in the myˉocardium of these patients and its course of progress with time.The present paper will attempt to discuss and anˉswer these questions through thetechnique of in situ hyˉbridization，using a cDNA Materials and Methods1Case selection.
1 Materials and Methods
1.1 Case selection Paraffin-embedded myocardial tissue samples of typical KDwere selected from65paˉtients who died of acute（12patients），subacute（33paˉtients）or chronic（20patients）KD.Control samples were collected from subjects who died without heart disˉease.The age，sex，onset date and endemic areas of these patients are shown in Table1.
Table1 Distribution of age，sex，date and areas of origin of clinical samplesTypes No. Age（No.）
Yunnan Province Abbreviations:No:Number of case;M:F，Male:Female;yr:years old.
1.2 Oligonucleotide probe An oligonucleotide probe was purchased from Shanghai Sango Biological Engineerˉing Company.This probe，labeled with biotin，was deˉsigned to detect a wide range of enterovirus，such as Coxsackievirus B3，B4，B5and A9（5′-CCT GTG GGT GGG TAC AAC CCA CAG GCT GTT-3′）.
1.3 Section preperation Five-micron sections were cut，placed on glass slides pretreated with APES，and stored at4℃.
1.4 In situ hybridization The paraffm wax was reˉmoved by immersing the slide in xylene.The slides were then rehydrated，treated with0.01%TritonX-100for2minutes，digested with proteinase K（0.25mg/ml）in PBS for5minutes at room temperature，sealed with hisˉtidine（2mg/ml）for2minutes，and then dehydrated in graded concentrated ethyl alcohol.The tissue sections were denatured on a80℃heating block for2minutes and cooled on ice immediately.They were pre-hyˉbridized for3～4h at42℃，by covering them with pre-hybridization liquid in wet chamber.After removal of the pre-hybridization liquid，the sections were allowed to hybridize overnight under the same conditions as deˉscribed above.
Following hybridization，the slides were rinsed twice with2X SSC containing50%deionized forˉmamide，twice with2X SSC，treated with0.1%Triton X-100for2minutes and washed twice in buffer I;Buffer II was added to each section and incubated for lh.After pouring off Buffer II，the SA-AP（streptaˉvidin，alkaline phosphatase conjugation）was added on to the tissue sections.They were allowed to react in this mixture for20minutes，rinsed with buffer I and then buffer IH.Finally，freshly prepared developer solution（BCIP/NBT）was applied to the slides and incubated for30min to4h.This produced a brownish blue color reaction.Following routine dehydration，the slides were cleared and mounted.
The positive hybridization signals of the Coxsackˉievirus RNA appeared as deep brownish blue mass or granules.In longitu