【摘要】目的探讨细胞毒T淋巴细胞(CTL)经Fas/FasL介导的细胞凋亡在柯萨奇病毒B₃ (CVB₃) 病毒性心肌炎(VM)小鼠发病中的作用。方法采用电镜技术及原位末端标记法（TUNEL）检测小鼠心肌组织的细胞凋亡，同时采用免疫组化、逆转录-聚合酶链反应（RT-PCR）及原位杂交3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（1）各期病鼠心肌组织中的TUNEL阳性的心肌细胞凋亡检出率为79%，中、重度VM小鼠的检出率明显高于轻度VM小鼠(P＜0.05)。平均心肌细胞凋亡百分率为（8±6）%。感染后第7～14天心肌细胞凋亡百分率明显高于第21～28天（P＜0.05）。 （2）感染后第7～14天,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（P＜0.01）。FasL蛋白表达水平与心肌病变积分呈正相关（r=0.89, P＜0.01）。结论细胞毒T淋巴细胞通过Fas/FasL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。

The role of Fas/FasL and apoptosis in the development of virus myocarditis in mice

HAN Bo, MA Peiran, LIU Fang, et al. Department of Pediatrics, Shandong Provincial Hospital, Jinan 250021, China

【Abstract】ObjectiveThe cytotoxic T lymphocyte (CTL) mediated cytotoxic reaction is one of the important pathogenesis of virus myocarditis (VM). CTL can induce apoptosis in cardiac muscle cells through Fas/Fas ligand (FasL) pathway. Apoptosis was proved exist in VM. The study, therefore, investigated the role of apoptosis and Fas/ FasL in the development of VM. MethodsOne hundred and twenty five Balb/c mice were included in the experiment. Twenty mice in experimental group inoculated with 10⁹ TCID Coxackie virus B₃(CVB₃) and five mice in control group injected with saline were sacrificed 7, 10, 14, 21 and 28 days post-inoculation (p.i.). Light microscopy, electronic microscopy and terminal transferase-mediated dUTP-biotin nick end-labeling (TUNEL) assays were used to detect the inflammation, necrosis and apoptosis in myocardium. 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) and in situ hybridization. Results(1) The incidence of VM was 86%. The typical myocardial lesions including myocardial necrosis and lymphocyte infiltration at the 7th day (p.i.) were observed, much more obviously at the 10th 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. (2)Apoptosis myocytes were seen in five of ten myocarditis mice by electronic microscopy. Apoptosis cells including myocytes, endothelial cells and infiltrating cells appeared in the myocardium of myocarditis mice. The detection rate of TUNEL-positive myocytes was 79%. The ratio of TUNEL-positive myocytes was much higher in mice with moderate to severe VM than mice with mild VM (P＜0.05). TUNEL-positive myocytes were mainly distributed in tela subendocardiaca, subepicardial layer and around the inflammatory foci. The average percent of apoptosis myocytes was (8±6)%. The percent of apoptosis myocytes increased significantly after the infection 7 to 14 days than 21 to 28 days post inoculation (P＜0.05). (3) In experimental group, Fas mRNA and protein expressed mainly in myocytes, and FasL mRNA and protein expressed mainly in infiltrating lymphocytes and increased remarkably from 7 to 14 days compared with control group(P＜0.01). The dynamic changes of FasL protein expression showed a significantly positive correlation with the changes of myocardial histopathologic scores (r=0.89, P＜0.01). ConclusionThe cytotoxic T lymphocyte mediated apoptosis in myocardium through Fas/FasL pathway might play an important role in the development of VM.

【Key words】Mice;Coxsackie viruses B;Myocarditis;T-lymphocytes;Apoptosis;Antigens, CD95

目前的研究表明，细胞毒T淋巴细胞（cytotoxic T lymphocyte ,CTL）介导的细胞毒作用是致病毒性心肌炎（viral myocarditis,VM）心肌细胞损伤的主要原因。Fas/FasL径路是CTL致靶细胞损伤的主要途径之一^［1］。VM存在心肌细胞凋亡，然而其分子调控机制尚未阐明。为此,本研究检测柯萨奇病毒B₃ (coxsackie virus B₃, CVB₃)所致 VM小鼠不同时期心肌组织中心肌细胞凋亡和Fas/FasL基因转录与蛋白表达水平，探讨CTL 经Fas/FasL介导的细胞凋亡在VM发病中的作用。

材料和方法

一、 CVB₃心肌炎动物模型的制备

由山东省医学科学院提供CVB₃病毒(Nancy株)。 125只4～6周龄雄性Balb/c小鼠由山东医科大学动物实验中心提供。随机将小鼠分成2组：实验组100只，每只腹腔接种0.12 ml含10⁹ TCID₅₀CVB₃的病毒液。对照组25只，腹腔接种等量生理盐水。分别于接种后第7、10、14、21、28天,随机取实验组小鼠20只、对照组小鼠5只，将其处死。取其心脏，半数冻存于-80℃冰箱，待做逆转录-聚合酶链反应（RT-PCR）及冰冻切片；半数用4％多聚甲醛固定，待做HE染色、免疫组化、TUNEL法检测，每批取2只病鼠的心肌用2.5％戊二醛固定，待做电镜检测。

二、病理学检查

常规HE染色，光镜下观察心肌病理变化，并计算心肌病变积分。即每张切片取5个视野,计算每个视野中炎性细胞浸润及坏死区域面积与整个视野的面积之比。无病变计0分,＜25%计1分,25%～50%计2分,50%～75%计3分,＞75%计4分。

三、TUNEL法原位检测心肌细胞凋亡

利用寡核苷酸末端脱氧核糖核酸转移酶（TdT）介导的dUTP缺口末端标记法检测。结果判定：随机计数5个高倍镜（×400）视野的心肌细胞总数及阳性心肌细胞数，按公式：阳性细胞/细胞总数×100％，计算心肌凋亡细胞百分率。

四、免疫组化检测Fas、FasL抗原

兔抗大鼠多克隆抗体Fas、FasL及SABC复合物均购自武汉博士德生物工程有限公司。按常规SABC法进行免疫组化检测，DAB染色，以PBS取代一抗和二抗作空白对照，光镜下观察结果。在MPIAS-500图像分析仪上，每个切片取5个高倍镜视野，测量心肌组织中阳性染色细胞的吸光度。

五、RT-PCR

采用Trizol(购自美国GBICO公司)提取总RNA，按AMV逆转录及PCR说明进行RT-PCR。引物序列：Fas： 5′-GACCCAGAATACCAAGTGCA-3′,5′-TCT-GTTCTGCTGTGTCTTGG-3′; FasL:5′-ATGGTTCTGG-TGGCTCTGGT-3′, 5′-GTTTAGGGGCTGGTTGTTGC-3′; 内参照基因β-actin： 5′-TGGAATCCTGTGGCATCC-ATGAAC-3′, 5′-TAAACGCAGCTCAGTAACAGTCCG-3′。可分别扩增436 bp的Fas cDNA、360 bp的FasL cDNA及349 bp的β-actin cDNA序列^［1,2］。扩增条件为94℃ 4 min, 94℃ 30 s,57℃ 30 s,72℃ 60 s,循环30次。在0.5 TBE电泳缓冲液中电压为 80 V电泳1 h,紫外灯下观察结果。并以PCR Marker作为标准分子量参照进行产物鉴定。用岛津-CS薄层扫描仪，测定PCR产物电泳条带的密度积分，计算Fas产物的相对量。计算公式：Fas相对量= Fas产物电泳条带密度/β-actin电泳条带密度×100%。

六、原位杂交

质粒来源（均由第二军医大学钱其军博士惠赠）：小鼠Fas cDNA载体质粒为pBluescript KS(+)，酶切位点为EcoR I。人FasL cDNA载体质粒为pBluescript-Ⅱ，酶切位点为Xbal-Ⅰ。按地高辛随机引物标记试剂盒（购自德国BM公司）说明书制备Fas cDNA、FasL cDNA探针，并按核酸检测试剂盒（购自德国BM公司）说明的步骤检测探针浓度。采用原位杂交检测心肌组织（冰冻切片）中Fas、FasL mRNA，以PBR322探针作为空白对照。图像分析同免疫组化。

七、统计学分析

计量资料用±s，采用PEMS统计软件包进行F-q′检验，计数资料应用精确卡方检验和秩和检验，部分指标间作直线相关分析，P＜0.05，差异具有显著性。

结果

一、CVB₃心肌炎小鼠心肌的病理变化

实验组小鼠于接种后第7天即见有明显的心肌细胞坏死和淋巴细胞浸润，第10～14天病变达高峰，第21天部分炎性病灶开始吸收，代之以纤维化，第28天多数病灶吸收。总感染率为86%，平均病变积分为(2.4±1.1)分。

二、CVB₃心肌炎小鼠心<