柯萨奇病毒RNA阳性信号在克山病心肌中形态特征及其与发病的关系
【摘要】 目的 探讨柯萨奇病毒RNA阳性信号在克山病心肌细胞中的形态特征及病毒感染与发病的关系。方法 以65例不同型别克山病尸检心肌组织蜡块为研究对象，用寡核苷酸探针与组织切片进行原位核酸杂交，以检测柯萨奇B组和A组病毒RNA。结果 急性、亚急性及慢性克山病的阳性杂交信号检出率分别为83.0%、87.9%及80.0%。阳性信号在急性、亚急性克山病心肌中颗粒粗大，境界较清晰，但分布比较局限;在慢性克山病中阳性信号多散在分布于各部位心肌细胞内，但颗粒较细小，境界不清。结论 心肌组织蜡块长期保存不影响杂交结果;吉林省各型克山病均与柯萨奇病毒感染密切相关，提示病毒感染是克山病复合病因的重要组成部分。
关键词 柯萨奇病毒 克山病 原位核酸杂交
【文献标识码】 A 【文章编号】 1606-8106(2004)12-1067-05
Morphological feature of positive hybridization signal
, http://www.100md.com
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
Engineering，Jilin University，Changchun130021.
【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.
, 百拇医药
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.
, http://www.100md.com
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.)
, 百拇医药
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℃.
, http://www.100md.com
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.
, http://www.100md.com
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.
, 百拇医药
2 Results
The positive hybridization signals of the Coxsackˉievirus RNA appeared as deep brownish blue mass or granules.In longitudinal section，they were seen to adˉhere to myofibrils，in one or several strings of beads(Fig.2).They varied insize and in cross section were usually localized in the sarcoplasm in the proximity of nuclei of myocytes，but no signals were found in the nuˉclei themselves(Fig.3).The color of signal was the darkest in the myocardium of papillary，muscles，trabecˉulae carneae and subendocardial muscle.It was lighter and less intense in the cells of the outer layers than those in the middle layers of ventricular walls.The myˉocardial cells surviving in fresh lesions and surrounding old necrotic foci often showed the hybridization signal.No signal was found in necrotic foci，and there were slight differences in the distribution and manifestation of positive hybrid signals in several types of KD.In most aˉcute and subacutelesions cases，the signal appeared preˉdominantly in the myocytes surroundingoci and survivˉing in fresh foci，as well as cells beneath the endocardiˉum and of papillary muscles.The hybridization-positive granules were thick，and had clear border.The sarˉcoplasm appeared dark，and sometimes，homogeneously blue(Fig.4).In most of the chronic cases，the granules were usually distributed in all parts of the myocardium，and were small and without clear border，like dirt(Fig.5).The detection rate in subacute specimens was the highest，and the rate inacute was higher than that in chronic，but there were no significant differences among the three types of KD.The details are shown in Table2.Table2.Positive hybridization rate of different type of KDAcute Subacute chronic No.of Hybridized 12 33 20 No.of Positive 10 29 16 Positive rate(%) 83.0 87.9 80.0
, 百拇医药
3 Discussion
3.1 Criteria for discriminating positive hybridization signals from nonspecific reactions，in addition to the posˉitive and negative controls，discrimination was also done between positive hybridization signals and nonspecific reactions in the normal structure of myocardium.Muscle fibrofilament presented in thin particulate form when muscles were cut transversely under microscope.In some abnormal conditions，fibrofilament thickening was alsoobserved，but the thin particulates were of the same size，and were distributed uniformly in the sarcoplasm.With positive signal，the difference consisted of varying sizes of granules localized around the nuclei.It was also very easy todistinguish positive signals from nonspecific reactions owing to higher unwinding temperatures.
, 百拇医药
We consider that the position，scope and distribuˉtion of signals were in accordance with the development of KD，and also coincided with the characteristics of host cell infection with the virus.Following the infection of the myocardium by the enterovirus，the viral RNA was translated into pre-ribosomal protein inthe ribosome in the smooth sarcoplasmic reticulum which accepted the viral RNA as mRNA [7] .Similar to other cells，smooth sarcoplasmic reticula of myocytes were distributed preˉdominantly around the nuclei，and on the outside of myˉofibril.Thus，positive hybridization signals were present in the sarcoplasma，most of them distributed in the area around the nuclei and appearing to stick onto themyˉofibril.All of the specimens used in this experiment were from paraffin-embedded autopsy tissues，kept for quite a long period，and some even over half a century.But all these sections showed clear signals by in situ hybridizaˉtion with oligonucleotide probe，demonstrating that proˉlonged storage did not affect the results of hybridization.In another words，Coxsackievirus RNA had not been fulˉly destroyed and did not disappear with time.
, http://www.100md.com
3.2 Changes in accumulation and decline of virus RNA in myocardium The presence of positive hyˉbridization signals indicates viral persistence in the myˉocardium of patients.The distribution characteristics of signal，relatively localized in acute and subacute KD but dispersed in chronic KD，suggesting that affected target cells increase in number in the course of persisting viral infection.Using athymic mice persistently infected with Coxsackievirus B3as a model systemand molecularly cloned Coxsackievims B3cDNA as a diagnostic probe，Kandolf et al [8] found that the viral RNA was located within the myocardium，the affected myocardium was disseminated and multifocal，and that it expanded from fibroticarea into uninfected area.This suggests the posˉsibility that viral infection proceeds from cell to cell.Further studies are needed to explain the mechanisms of this expansion，its influence on the metabolism and funcˉtion of the myocardium，and the relationship between this expansion and clinical course of chronic KD.
, 百拇医药
The fact that there were no positive signals in necrotic and fibrotic foci showed that the viral RNA was destroyed and disappeared following necrosis and dissoˉlution of myocytes.But our concern was as to howthe viˉral RNA interacts with target cells in the living myˉocardium and whether the target cells had thecapacity to restrict virus duplication and expansion.In the present experiments，the positive granules in the specimens of chronic KD were thin，pale in color，and unclear border，which appeared to implicate that the myocardium of host might be able to restrict virus duplication.This deserves further investigation.
, 百拇医药
3.3 Correlation between enterovirus infection and ocˉcurrence of KD.Some cases of subacute KD，which ocˉcured predominantly in children in the Southwestern disˉtricts of China and was prevail mainly in summer and autumn，were accompanied by abdominal pain，diarrhoea or symptoms of respiratory tract infection.A large numˉbers of autopsy cases were also revealed the presence of interstitial inflammation in the lung，pancreas，kidney，liver，mesentery lymph node，and other tissues.Early on the authors considered the possibility of viral infection in these cases [9，10] .But the authors were uncertain of whether those KD cases，such as acute and severe cases occurred mainly toward the end of1950s and the early days of1960s in the Jilin province，were related to enˉteroviral infection.These patients were scattered in the remote mountainous villages in deep winter，and their clinical course and epidemiological characteristics did not conform tothose generally seen in enteroviral infecˉtion.Also，there was no clear pathological evidence of viˉral infection.As our results of in situ hybridization shows not only positive signals in the myocardium of these casˉes，but also the existence of numerous thick positive grantfies with clear border，indicating extensive virus duˉplication within the myocardium.This confirms our hyˉpothesis that enterovims is an important component of the multiple causality of KD [11] .
, 百拇医药
Selenium and vitamin E deficiencies may be pathogenic factors which contribute to regional distribution of KD.The variable association of Se and VE deficiencieswith enteroviral infection and other environmental factors may decide the variety of clinical and epidemiological characteristics of KT.Perˉhaps the continueddecrease in the incidence of KD，which has even gradually disappeared in some epidemic areas since the late1970s，is the result of a break in the complex web of its causation factors(for example，imˉprovement in nutritional conditions and increased dietary intake of vitamin E，sulfhydryl-containing amino acidsas well as selenium) [12] . Beck，Levander et al [13～19] found thatthe genome of Coxsackievirus B3could be changed in selenium or vitamin E-deficient mice the normal benign(amyˉocarditic)strain became virulent.In our futurestudies，we hope to sequence the Coxsackieviral genome isolated from heart samples of the patients who died of KD，in orˉder to detect the presence of any mutations，so as to furˉther define the exact interrelation between Se-deficienˉcy and viral infection in the multiple causality of KD. (本文图片见附页)
, 百拇医药
参考文献
1 Li GS，Wang F，Kang DR.Keshan disease:an endemic cardiomyopathy in China.Hum Pathol，1985，16:602-609.
2 Zhong XK，Zhang FM，Jiang ZR，et al.Relationship between Coxsackˉievirus B3and subacute Keshan disease in Yunnan province by in situ hybridization.Chin J Ctrl Endem Dis，1993，12(4):193-195.
3 Niu MJ，Zhong XK，Jiang ZR，et al.Relationship between Coxsackˉievirus B3and chronic Keshan disease in Yunnan province by in situ hybridization.Chin J Ctrl Endem Dis，1996，15(2):65-67.
, 百拇医药
4 Zhong XK，Zhou N，Niu MJ，et al.Relationship between Coxsackie B virus andKeshan disease by in situ hybridization(acute Kenshan disease in Heilongjiang province).Chin J Ctrl Endem Dis，1995，14(6):323-325.
5 Zhong XK，Niu MJ，Yang XH，et al.Relationship between Coxsackie B virus and chronic Keshan disease by in situ hybridization.Chin J Ctrl Endem Dis，1996，15(1):8-9.
6 Zhou LW，Zhong XK，Niu MJ，et al.Relationship between Coxsackˉievirus B and chronic Keshan disease in Heilongjiang province by in situ hybridization.ChinJ Ctrl Endem Dis，1996，15(2):65-67.
, 百拇医药
7 Peng TQ，Li YW，Yang YZ，et al.Characterization of enterovirus isolatˉed from patients with heart muscle disease in a selenium-deficient area of China.J Clin Microbiol，2000，38(10):3538-3543.
8 Kandolf R，Klingel K，Zell R，et al.Molecular mechanisms in the pathoˉgenesis of enteroviral heart disease:acute and persistent infections.Clin Immunol Immunopathol，1993，68(2):153-158.
9 Li GS，Wang F，Lou ZP，et al.Pathological research on children Keshan disease in northeast/southwest of China.J Jilin Med Universe，1976，3(2):117-136.
, 百拇医药
10 Li GS，Yang GF.Morphologic feature of children Kenshan disease conˉcur infection.Chin J Ctrl Endem Dis，1984，3(1):3-8.
11 Su Y，Yu WH.Nutritional biochemical etiology of Keshan disease.Clin Med J，1983，96:594-596.
12 Li GS，Wang Fan.Pathogenesis of Keshan disease and the basic underˉstandto pathogeny:A Review.Li GS.Selenium deficiency and its relaˉtion factor in Keshan Disease.Chang Chun:Science and Technology Publishing House of Jilin Provice，1997，247-266.
, http://www.100md.com
13 BeckMA，Kolbeck PC，Rohr LH，et al.Vitamin E deficiency intersifies the myocardial injury of Coxsackievirus B infection of mice.J Nutr，1994，124:345-358.
14 Beck MA，Kolbeck PC，Shi Q，et al.Increased virulence of a human enterovirus(Coxsackievirus B3)in selenium-deficient mice.J Infect Dis，1994，170:351-357.
15 Beck MA，Kolbeck PC，Rohr L，et al.Benign human enterovirus beˉcomes virulent in selenium-deficient mice.J Med5rol，1994，43:166-170.
, http://www.100md.com
16 BeckMA，Shi Q，Morris VC，et al.Rapid genomic evolution of a non-virulent Coxsackievirus B3in selenium-deficient mice results in seˉlection of identical virulent isolates.Nature Medicine，1995，1:433-436.
17 Beck MA.Rapid genomic evolution of a non-virulent CoxsackievirusB3in selenium-deficient mice.Biomed Environ Sci，1997，10:307- 315.
18 Levander OA，Beck MA.Interacting nutritional and infectious etioloˉgies of Keshan disease.Biol Trace Elem Res，1997，56:5-21.
19 Levander OA.The selenium-Coxsackievirus connection:Chronicle of a collaboration.J Nutr，2000，130:485-488.
作者单位:130021吉林长春吉林大学地方病研究所
吉林大学第一医院心外科
(编辑李 木), 百拇医药
关键词 柯萨奇病毒 克山病 原位核酸杂交
【文献标识码】 A 【文章编号】 1606-8106(2004)12-1067-05
Morphological feature of positive hybridization signal
, http://www.100md.com
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
Engineering，Jilin University，Changchun130021.
【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.
, 百拇医药
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.
, http://www.100md.com
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.)
, 百拇医药
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℃.
, http://www.100md.com
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.
, http://www.100md.com
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.
, 百拇医药
2 Results
The positive hybridization signals of the Coxsackˉievirus RNA appeared as deep brownish blue mass or granules.In longitudinal section，they were seen to adˉhere to myofibrils，in one or several strings of beads(Fig.2).They varied insize and in cross section were usually localized in the sarcoplasm in the proximity of nuclei of myocytes，but no signals were found in the nuˉclei themselves(Fig.3).The color of signal was the darkest in the myocardium of papillary，muscles，trabecˉulae carneae and subendocardial muscle.It was lighter and less intense in the cells of the outer layers than those in the middle layers of ventricular walls.The myˉocardial cells surviving in fresh lesions and surrounding old necrotic foci often showed the hybridization signal.No signal was found in necrotic foci，and there were slight differences in the distribution and manifestation of positive hybrid signals in several types of KD.In most aˉcute and subacutelesions cases，the signal appeared preˉdominantly in the myocytes surroundingoci and survivˉing in fresh foci，as well as cells beneath the endocardiˉum and of papillary muscles.The hybridization-positive granules were thick，and had clear border.The sarˉcoplasm appeared dark，and sometimes，homogeneously blue(Fig.4).In most of the chronic cases，the granules were usually distributed in all parts of the myocardium，and were small and without clear border，like dirt(Fig.5).The detection rate in subacute specimens was the highest，and the rate inacute was higher than that in chronic，but there were no significant differences among the three types of KD.The details are shown in Table2.Table2.Positive hybridization rate of different type of KDAcute Subacute chronic No.of Hybridized 12 33 20 No.of Positive 10 29 16 Positive rate(%) 83.0 87.9 80.0
, 百拇医药
3 Discussion
3.1 Criteria for discriminating positive hybridization signals from nonspecific reactions，in addition to the posˉitive and negative controls，discrimination was also done between positive hybridization signals and nonspecific reactions in the normal structure of myocardium.Muscle fibrofilament presented in thin particulate form when muscles were cut transversely under microscope.In some abnormal conditions，fibrofilament thickening was alsoobserved，but the thin particulates were of the same size，and were distributed uniformly in the sarcoplasm.With positive signal，the difference consisted of varying sizes of granules localized around the nuclei.It was also very easy todistinguish positive signals from nonspecific reactions owing to higher unwinding temperatures.
, 百拇医药
We consider that the position，scope and distribuˉtion of signals were in accordance with the development of KD，and also coincided with the characteristics of host cell infection with the virus.Following the infection of the myocardium by the enterovirus，the viral RNA was translated into pre-ribosomal protein inthe ribosome in the smooth sarcoplasmic reticulum which accepted the viral RNA as mRNA [7] .Similar to other cells，smooth sarcoplasmic reticula of myocytes were distributed preˉdominantly around the nuclei，and on the outside of myˉofibril.Thus，positive hybridization signals were present in the sarcoplasma，most of them distributed in the area around the nuclei and appearing to stick onto themyˉofibril.All of the specimens used in this experiment were from paraffin-embedded autopsy tissues，kept for quite a long period，and some even over half a century.But all these sections showed clear signals by in situ hybridizaˉtion with oligonucleotide probe，demonstrating that proˉlonged storage did not affect the results of hybridization.In another words，Coxsackievirus RNA had not been fulˉly destroyed and did not disappear with time.
, http://www.100md.com
3.2 Changes in accumulation and decline of virus RNA in myocardium The presence of positive hyˉbridization signals indicates viral persistence in the myˉocardium of patients.The distribution characteristics of signal，relatively localized in acute and subacute KD but dispersed in chronic KD，suggesting that affected target cells increase in number in the course of persisting viral infection.Using athymic mice persistently infected with Coxsackievirus B3as a model systemand molecularly cloned Coxsackievims B3cDNA as a diagnostic probe，Kandolf et al [8] found that the viral RNA was located within the myocardium，the affected myocardium was disseminated and multifocal，and that it expanded from fibroticarea into uninfected area.This suggests the posˉsibility that viral infection proceeds from cell to cell.Further studies are needed to explain the mechanisms of this expansion，its influence on the metabolism and funcˉtion of the myocardium，and the relationship between this expansion and clinical course of chronic KD.
, 百拇医药
The fact that there were no positive signals in necrotic and fibrotic foci showed that the viral RNA was destroyed and disappeared following necrosis and dissoˉlution of myocytes.But our concern was as to howthe viˉral RNA interacts with target cells in the living myˉocardium and whether the target cells had thecapacity to restrict virus duplication and expansion.In the present experiments，the positive granules in the specimens of chronic KD were thin，pale in color，and unclear border，which appeared to implicate that the myocardium of host might be able to restrict virus duplication.This deserves further investigation.
, 百拇医药
3.3 Correlation between enterovirus infection and ocˉcurrence of KD.Some cases of subacute KD，which ocˉcured predominantly in children in the Southwestern disˉtricts of China and was prevail mainly in summer and autumn，were accompanied by abdominal pain，diarrhoea or symptoms of respiratory tract infection.A large numˉbers of autopsy cases were also revealed the presence of interstitial inflammation in the lung，pancreas，kidney，liver，mesentery lymph node，and other tissues.Early on the authors considered the possibility of viral infection in these cases [9，10] .But the authors were uncertain of whether those KD cases，such as acute and severe cases occurred mainly toward the end of1950s and the early days of1960s in the Jilin province，were related to enˉteroviral infection.These patients were scattered in the remote mountainous villages in deep winter，and their clinical course and epidemiological characteristics did not conform tothose generally seen in enteroviral infecˉtion.Also，there was no clear pathological evidence of viˉral infection.As our results of in situ hybridization shows not only positive signals in the myocardium of these casˉes，but also the existence of numerous thick positive grantfies with clear border，indicating extensive virus duˉplication within the myocardium.This confirms our hyˉpothesis that enterovims is an important component of the multiple causality of KD [11] .
, 百拇医药
Selenium and vitamin E deficiencies may be pathogenic factors which contribute to regional distribution of KD.The variable association of Se and VE deficiencieswith enteroviral infection and other environmental factors may decide the variety of clinical and epidemiological characteristics of KT.Perˉhaps the continueddecrease in the incidence of KD，which has even gradually disappeared in some epidemic areas since the late1970s，is the result of a break in the complex web of its causation factors(for example，imˉprovement in nutritional conditions and increased dietary intake of vitamin E，sulfhydryl-containing amino acidsas well as selenium) [12] . Beck，Levander et al [13～19] found thatthe genome of Coxsackievirus B3could be changed in selenium or vitamin E-deficient mice the normal benign(amyˉocarditic)strain became virulent.In our futurestudies，we hope to sequence the Coxsackieviral genome isolated from heart samples of the patients who died of KD，in orˉder to detect the presence of any mutations，so as to furˉther define the exact interrelation between Se-deficienˉcy and viral infection in the multiple causality of KD. (本文图片见附页)
, 百拇医药
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, 百拇医药
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, 百拇医药
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, 百拇医药
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, http://www.100md.com
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作者单位:130021吉林长春吉林大学地方病研究所
吉林大学第一医院心外科
(编辑李 木), 百拇医药
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