小鼠病毒性心肌炎中心肌损伤及其与Fas/FasL异常表达的关系的研究
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摘要
目的：探讨柯萨奇病毒B_3(CVB_3)所致小鼠病毒性心肌炎(VM)心肌细胞损伤的变化，观察经Fas/FasL介导的细胞凋亡在心肌损伤中的作用。
方法：采用CVB_3制备小鼠病毒性心肌炎动物模型，分别于腹腔接种CVB_3后第5、7、10、14、21、28天，取外周血清检测肌酸磷酸激酶(CK)、心肌组织切片HE染色了解心肌损伤(坏死)情况；流式细胞仪检测和电镜检测，观察心肌中的细胞凋亡；同时采用免疫组化、逆转录-聚合酶链反应(RT-PCR)两种方法检测心肌炎小鼠不同时期心肌组织中Fas与FasL基因转录与蛋白的表达。
结果：心肌坏死方面：光镜观察见对照组小鼠心肌组织无异常改变；实验组小鼠心肌细胞初时肿胀，细胞横纹不清，胞质染色嗜酸性增强，胞核出现核固缩及核碎裂，而间质一般无炎性细胞浸润，其后变性心肌进一步坏死崩解，胞核和细胞轮廓消失，周围出现炎性细胞浸润(主要是单核细胞和淋巴细胞)，间质内可见较多的胶原纤维；外周血清CK检测于病毒接种后第5天即有所升高，7～14天较对照组明显增加，其后有所降低，至28天时接近正常，但其变化并不出现如急性心肌梗死的动态变化，并且可以持续相当长时间。心肌细胞凋亡方面：电镜观察对照组心肌中未检出凋亡细胞，而病鼠心肌凋亡细胞检出率较高58.33％(7/12例)，且细胞凋亡多存在于炎症病灶周围的心肌细胞及病灶处炎性浸润细胞；各期病鼠心肌组织中的细胞凋亡比率较对照组明显增加，流式细胞仪检测平均心肌细胞凋亡百分率为26.74±15.22％，感染后第7～14天心肌细胞凋亡百分率明显高
于第5大及21、28天O＜0刀01人 感染后第7～14天的病鼠心肌组织中，
主要表达于心肌细胞的Fas mRNA及蛋白和主要表达于浸润淋巴细胞的
FasL mRNA及蛋白均明显增强；FasL蛋白表达水平与心肌病变积分呈正相
关（＊o．9082，P＜0刀01）。
结论：小鼠病毒性心肌炎中。0肌损伤坏死与凋亡共存，通过FasN肪L
基因径路介导的心肌细胞凋亡与病毒性心肌炎的发病过程有关。
Objectives: The mechanisms responsible for myocardial injury and cell death in myocarditis are still unclear. Necrosis and apoptosis were proved exist in virus myocarditis (VM). Fas/FasL belong to the tumor necrosis factor receptor/ligand superfamily of costimulatory molecules and are known to play a critical role in the induction of apoptosis, as well as in the cytotoxicty mediated by T-cells and natural killer cells. In this study, we examined that myocardial cell death occurs in murine acute myocarditis caused by Coxsackie virus B3 (CVB3) and whether the Fas/Fas ligand (FasL) system plays a role in myocardial damage(especially in the myocytes apoptosis) and the development of VM .
Methods: One hundred and thirty BALB/c mice were included in the experiment. Fifteen mice in experimental group inoculated with CVB3 and five mice in control group injected with Eagle's minimum essential medium (MEM) were sacrificed 5, 7, 10, 14, 21, 28 days post-inoculation (p.i.). Light microscopy, electronic microscopy and flow cytometry (FCM) were used to detect the inflammation, necrosis and apoptosis in myocardium. Serum creatine phosphokinase (CK) measurements were performed as marker enzymes of the mice myocytes lesion progression induced by CVB3 infection. The expressions of Fas and FasL protein in myocardium were determined by immunohistochemistry. Fas mRNA and FasL mRNA were analyzed by reverse-transcription polymerase chain reaction (RT-PCR).
Results: (1) The typical myocardial lesions including myocardial necrosis and lymphocyte infiltration at the 5th day p.i. were observed, much more obviously at the 7th to 14th day, and recovered gradually from the 21th to 28th day p.i. The mice in control group did not show any sign of inflammation. The level of serum CK increased at the 5th day p.i., much more obviously at the 7th to 14th day p.i., and recovered gradually from the 21th to 28th day p.i. But the level of serum CK in mice of VM did not increase as it changed in acute myocardial infarction. (2) Apoptosis myocytes were seen in seven of twelve myocarditis mice by electronic microscopy. Apoptosis cells including myocytes (around the nidus) and infiltrating lymphocytes were detected in the myocardium of myocarditis mice by electronic microscopy.But apoptosis cells were not observed in the mice of control group. (3) The detection rate apoptosis cells in myocardium was much higher in mice with VM than the mice
in control group . The percent of apoptosis cells increased significantly after the infection 7 to 14 days than 5 p.i.and 21 to 28 days p.i. (4) In experimental group, Fas protein expressed mainly in myocytes and FasL protein expressed mainly in infiltrating lymphocytes and increased remarkably from 7 to 14 days compared with control group(P<0.001) . The dynamic changes of FasL protein expression showed a significantly positive correlation with the changes of myocardial histopathologic scores (r= 0.9082, P<0.001) . Fas mRNA in experimental group increased remarkably from 7 to 14 days p.i. compared with control group(P<0.00l).
Conclusion: These findings suggest that cell death via necrosis and apoptosis existed in murine acute myocarditis caused by CVBs at the same time, apoptosis of cardiomyocytes and lymphocytes is one of the mechanisms of myocardial injury in VM, and that the cytotoxic T lymphocyte mediatied myocardium damage through Fas/FasL pathway might play an important role in the development of VM.
方法：采用CVB_3制备小鼠病毒性心肌炎动物模型，分别于腹腔接种CVB_3后第5、7、10、14、21、28天，取外周血清检测肌酸磷酸激酶(CK)、心肌组织切片HE染色了解心肌损伤(坏死)情况；流式细胞仪检测和电镜检测，观察心肌中的细胞凋亡；同时采用免疫组化、逆转录-聚合酶链反应(RT-PCR)两种方法检测心肌炎小鼠不同时期心肌组织中Fas与FasL基因转录与蛋白的表达。
结果：心肌坏死方面：光镜观察见对照组小鼠心肌组织无异常改变；实验组小鼠心肌细胞初时肿胀，细胞横纹不清，胞质染色嗜酸性增强，胞核出现核固缩及核碎裂，而间质一般无炎性细胞浸润，其后变性心肌进一步坏死崩解，胞核和细胞轮廓消失，周围出现炎性细胞浸润(主要是单核细胞和淋巴细胞)，间质内可见较多的胶原纤维；外周血清CK检测于病毒接种后第5天即有所升高，7～14天较对照组明显增加，其后有所降低，至28天时接近正常，但其变化并不出现如急性心肌梗死的动态变化，并且可以持续相当长时间。心肌细胞凋亡方面：电镜观察对照组心肌中未检出凋亡细胞，而病鼠心肌凋亡细胞检出率较高58.33％(7/12例)，且细胞凋亡多存在于炎症病灶周围的心肌细胞及病灶处炎性浸润细胞；各期病鼠心肌组织中的细胞凋亡比率较对照组明显增加，流式细胞仪检测平均心肌细胞凋亡百分率为26.74±15.22％，感染后第7～14天心肌细胞凋亡百分率明显高
于第5大及21、28天O＜0刀01人 感染后第7～14天的病鼠心肌组织中，
主要表达于心肌细胞的Fas mRNA及蛋白和主要表达于浸润淋巴细胞的
FasL mRNA及蛋白均明显增强；FasL蛋白表达水平与心肌病变积分呈正相
关（＊o．9082，P＜0刀01）。
结论：小鼠病毒性心肌炎中。0肌损伤坏死与凋亡共存，通过FasN肪L
基因径路介导的心肌细胞凋亡与病毒性心肌炎的发病过程有关。
Objectives: The mechanisms responsible for myocardial injury and cell death in myocarditis are still unclear. Necrosis and apoptosis were proved exist in virus myocarditis (VM). Fas/FasL belong to the tumor necrosis factor receptor/ligand superfamily of costimulatory molecules and are known to play a critical role in the induction of apoptosis, as well as in the cytotoxicty mediated by T-cells and natural killer cells. In this study, we examined that myocardial cell death occurs in murine acute myocarditis caused by Coxsackie virus B3 (CVB3) and whether the Fas/Fas ligand (FasL) system plays a role in myocardial damage(especially in the myocytes apoptosis) and the development of VM .
Methods: One hundred and thirty BALB/c mice were included in the experiment. Fifteen mice in experimental group inoculated with CVB3 and five mice in control group injected with Eagle's minimum essential medium (MEM) were sacrificed 5, 7, 10, 14, 21, 28 days post-inoculation (p.i.). Light microscopy, electronic microscopy and flow cytometry (FCM) were used to detect the inflammation, necrosis and apoptosis in myocardium. Serum creatine phosphokinase (CK) measurements were performed as marker enzymes of the mice myocytes lesion progression induced by CVB3 infection. The expressions of Fas and FasL protein in myocardium were determined by immunohistochemistry. Fas mRNA and FasL mRNA were analyzed by reverse-transcription polymerase chain reaction (RT-PCR).
Results: (1) The typical myocardial lesions including myocardial necrosis and lymphocyte infiltration at the 5th day p.i. were observed, much more obviously at the 7th to 14th day, and recovered gradually from the 21th to 28th day p.i. The mice in control group did not show any sign of inflammation. The level of serum CK increased at the 5th day p.i., much more obviously at the 7th to 14th day p.i., and recovered gradually from the 21th to 28th day p.i. But the level of serum CK in mice of VM did not increase as it changed in acute myocardial infarction. (2) Apoptosis myocytes were seen in seven of twelve myocarditis mice by electronic microscopy. Apoptosis cells including myocytes (around the nidus) and infiltrating lymphocytes were detected in the myocardium of myocarditis mice by electronic microscopy.But apoptosis cells were not observed in the mice of control group. (3) The detection rate apoptosis cells in myocardium was much higher in mice with VM than the mice
in control group . The percent of apoptosis cells increased significantly after the infection 7 to 14 days than 5 p.i.and 21 to 28 days p.i. (4) In experimental group, Fas protein expressed mainly in myocytes and FasL protein expressed mainly in infiltrating lymphocytes and increased remarkably from 7 to 14 days compared with control group(P<0.001) . The dynamic changes of FasL protein expression showed a significantly positive correlation with the changes of myocardial histopathologic scores (r= 0.9082, P<0.001) . Fas mRNA in experimental group increased remarkably from 7 to 14 days p.i. compared with control group(P<0.00l).
Conclusion: These findings suggest that cell death via necrosis and apoptosis existed in murine acute myocarditis caused by CVBs at the same time, apoptosis of cardiomyocytes and lymphocytes is one of the mechanisms of myocardial injury in VM, and that the cytotoxic T lymphocyte mediatied myocardium damage through Fas/FasL pathway might play an important role in the development of VM.
引文
1 张旭晨。心血管疾病与细胞凋亡。国外医学 生理·病理科学与临床分册，1998；18：335-337．
2 Kawano H, Okada R, Kauano Y, et al.Apoptosis in acute and chronic myocarditis. Jpn Heart J, 1994,35:745-750.
3 Toyozaki T, Hiroe M, Tanaka M. Levels of soluble Fas ligand in myocarditis.Am J Cardio, 1998,82:246-248.
4 Huber SA.Coxsackievirus-induced myocarditis is dependent on distinct immunopathogenic responses in different strains of mice.Lab Invest, 1997,76:691-701.
5 Chocron S,Alwan W, Toubin G, et al. Effects of myocardial ischemia on the release of cardic troponin Ⅰ in isolated rat hearts [J].J Thorac Cardiovasc Surg 1996,112:508.
6 朱健华，姚登福。心肌损伤早期血液Troponin Ⅰ及酶学标志的动态变化。天津医药，2000；29(4)：223-226。
7 Akkerhuis KM, Alexander JH, Tardiff BE,et al.Minor myocardial damage and prognosis: are spontaneous and percutaneous coronary intervention-related events different? Circulation 2002; 105(5):554-6
8 Brener SJ, Lytle BW, Schneider JP, et al. Association between CK-MB elevation after percutaneous or surgical revascularization and three-year mortality. J Am Coll Cardiol 2002 Dec 4;40(11): 1961-7
9 Debiasi RL, Edelstein CL, Sherry B, et al. Calpain inhibition protects against virus-induce myocardial injury. J Virol, 2001, 75(1): 351～361
10 Colston JT, Chandrasekar B, Freeman GL. Expression of apoptosis-related proteins in experimental coxsackievirus myocarditis. Cardiovasc Res. 1998 Apr;38(1): 158-68.
11 Huber SA. Coxsackievirus induced myocarditis is dependent on distinct
immunopathogenic response in different strain of mice. Lab Invest, 1997; 76(5): 691
12 Huber SA. T cells expressing the gamma delta T cell receptor induce apoptosis in cardiac myocytes. Cardiovasc Res 2000;45(3):579-87
13 Gebhard IR, Perry CM, Harkins S, et al. The role of CD8~+ T lymphocytes in coxsackievirus B3-induced myocarditis: perforin exacerbates disease, but play no detectable role in virus clearance. Am J Pathol, 1998,153(2):417～428.
2 Kawano H, Okada R, Kauano Y, et al.Apoptosis in acute and chronic myocarditis. Jpn Heart J, 1994,35:745-750.
3 Toyozaki T, Hiroe M, Tanaka M. Levels of soluble Fas ligand in myocarditis.Am J Cardio, 1998,82:246-248.
4 Huber SA.Coxsackievirus-induced myocarditis is dependent on distinct immunopathogenic responses in different strains of mice.Lab Invest, 1997,76:691-701.
5 Chocron S,Alwan W, Toubin G, et al. Effects of myocardial ischemia on the release of cardic troponin Ⅰ in isolated rat hearts [J].J Thorac Cardiovasc Surg 1996,112:508.
6 朱健华，姚登福。心肌损伤早期血液Troponin Ⅰ及酶学标志的动态变化。天津医药，2000；29(4)：223-226。
7 Akkerhuis KM, Alexander JH, Tardiff BE,et al.Minor myocardial damage and prognosis: are spontaneous and percutaneous coronary intervention-related events different? Circulation 2002; 105(5):554-6
8 Brener SJ, Lytle BW, Schneider JP, et al. Association between CK-MB elevation after percutaneous or surgical revascularization and three-year mortality. J Am Coll Cardiol 2002 Dec 4;40(11): 1961-7
9 Debiasi RL, Edelstein CL, Sherry B, et al. Calpain inhibition protects against virus-induce myocardial injury. J Virol, 2001, 75(1): 351～361
10 Colston JT, Chandrasekar B, Freeman GL. Expression of apoptosis-related proteins in experimental coxsackievirus myocarditis. Cardiovasc Res. 1998 Apr;38(1): 158-68.
11 Huber SA. Coxsackievirus induced myocarditis is dependent on distinct
immunopathogenic response in different strain of mice. Lab Invest, 1997; 76(5): 691
12 Huber SA. T cells expressing the gamma delta T cell receptor induce apoptosis in cardiac myocytes. Cardiovasc Res 2000;45(3):579-87
13 Gebhard IR, Perry CM, Harkins S, et al. The role of CD8~+ T lymphocytes in coxsackievirus B3-induced myocarditis: perforin exacerbates disease, but play no detectable role in virus clearance. Am J Pathol, 1998,153(2):417～428.