风湿性心脏病心肌中增殖细胞核抗原和α-平滑肌肌动蛋白的表达与心功能关系.doc

风湿性心脏病心肌中增殖细胞核抗原和-平滑肌肌动蛋白的表达与心功能关系风湿性心脏病心肌中增殖细胞核抗原和-平滑肌肌动蛋白的表达与心功能关系2010-09-07 张近宝刘维永晏培松王剑波岳瑾琢朱霆 【摘要】目的阐明慢性风湿性心脏病(风心病)心肌中增殖细胞核抗原（PCNA）、-平滑肌肌动蛋白（-SMA）阳性表达的意义及与心功能的关系。方法用PCNA、-SMA单克隆抗体对风心病二尖瓣病变心肌组织进行免疫组化及免疫电镜观察，图像分析仪定量分析其阳性表达率、心肌纤维化面积、微动脉内外径比值,并与左室心功能对比。结果发现风心病心肌间质纤维化和微动脉管壁增厚,管腔变窄。PCNA在风心病肥大心肌细胞和间质细胞核中均有表达、-SMA在风心病心肌间质细胞中有表达，后者多位于血管周边。结论（1）风心病心肌细胞表型发生改变,PCNA的阳性表达率与心肌纤维化面积成正比；（2）间质细胞不仅参与了心肌间质纤维化,且在血管改建中起重要作用；（3）PCNA阳性表达程度及心肌纤维化面积与左心功能成负相关。 【关键词】风湿性心脏病增殖细胞核抗原肌动蛋白类心室功能 Study of the relationship between expression of PCNA, -SMA and left ventricle cardiac function in myocardium of the patients with rheumatic heart diseaseZhang Jinbao, Liu Weiyong, Yan Peisong, et al. Xijing Hospital Fourth Military Medical University, PLA, Xian, 710032 【Abstract】ObjectiveTo reveal the significance of positive expression of PCNA, -SMA and the relationship between that and left ventricular cardiac function in myocardium of the patients with rheumatic heart disease. MethodsThe papillary muscle samples obtained from the patients with rheumatic heart disease were immunohistochemically stained with monoclonal antibody of PCNA, -SMA and observed by immunoelectron microscopy. Ima-ging analysis was employed to measure the degree of immunohistochemical staining, the areas of myocardium fibrosis and the ratios between arteriolar inner and outer diameter. The data were compared with cardiac function.ResultsInterstitial fibrosis in rheumatic myocardium was significant. The wall of small arteries appeared thick and tune appeared narrow. Immunohistochemical PCNA positive expression was exhibited in hypertrophic cardiomyocytes and interstitial cell nuclei. -SMA positive expression was in interstitial cells of rheumatic myocardium, and most of these cells appeared around the artery.Conclusion(1)The phenotype of myocardial cell had altered, the rate of PCNA positive expression was directly proportional to the areas of myocardium fibrosis. (2) Interstitial cells not only take part in interstitial fibrosis progress but also play a key role in vascular reconstruction. (3)The degree of PCNA positive expression and the areas of myocardium fibrosis were negative associated with cardiac function. 【Key words】rheumatic heart diseaseproliferating cell nuclear antigenactinsventricular function 增殖细胞核抗原(PCNA)是真核细胞DNA合成所必须的一种核蛋白，近年来被用作原位检测细胞增殖状态的特异性抗体而用于肿瘤动力学研究1。有学者报道未成熟心肌和晚期衰竭心肌中有PCNA阳性表达2,3。-平滑肌肌动蛋白(-SMA)是一种细胞骨架蛋白,正常人心肌仅血管平滑肌细胞呈阳性表达。最近有报道肌纤维母细胞(MFb)也可表达-SMA4,而MFb与器官纤维化关系密切,但与心肌纤维化的关系未见报道。本研究应用免疫组化及免疫电镜发现在风湿性心脏病（风心病）心肌组织中PCNA呈阳性表达、-SMA在心肌间质中呈阳性表达,并将其表达率与手术前后左室心功能变化进行对比分析,以探讨心肌病变对心功能的影响。 资料与方法 1.材料来源: 38例病变标本选自我院1995年1月至1995年12月确诊为风心病、单纯二尖瓣病变,行二尖瓣替换术患者左室乳头肌（除外合并有主动脉瓣病变或合并有冠心病者）,其中男17例,女21例（年龄为39.79.4岁)。心功能级26例,级12例,病史530年。术前按二尖瓣瓣口面积及反流程度分为：（1）二尖瓣狭窄组16例（瓣口面积1.2cm2,反流量1.5cm2,反流量6ml)，两组患者一般情况无统计学差异；（3）对照组(5例):取自意外死亡而无心肺疾病男性的左室乳头肌,年龄在2248岁；（4）胎儿尸检标本对照组(5例):取自不同月龄的死胎左室乳头肌,月龄在37个月。鼠抗人-SMA单克隆抗体为Sigma公司产品,鼠抗人PCNA单克隆抗体及S-P免疫组化试剂盒为Dako公司产品。 2.标本制备:病变组在手术中取材,对照组正常成年及胎儿乳头肌标本均在死后半小时内取材,标本被分成2份，1份立即放入10福尔马林液中固定,常规石蜡包埋,连续切片厚3m,分别作HE及免疫组化染色,另一份用含4多聚甲醛和0.05%戊二醛的饱和苦味酸液中浸泡5小时,振动切片厚30m,作包埋前漂浮染色。 3.检测指标和方法:(1)所有患者均于手术前和手术后6个月内应用二维超声心动图和彩色多普勒分别检查,测量左室各项心功能指标,包括左室心肌重量;(2)病理标本做免疫组化染色及免疫电镜观察。免疫组化染色及免疫电镜染色按SP法（链霉素亲生物蛋白-过氧化酶连接法）进行。电镜标本染色后常规经树酯包埋，高温聚合，光镜下取阳性部位作超薄切片，经醋酸双氧铀及柠檬酸铅双重电子染色后电镜下观察。 4.结果判定和记录: PCNA阳性以肝癌细胞为阳性对照,定位于细胞核;-SMA以血管壁平滑肌细胞作阳性对照定位于胞浆。将免疫组化及HE染色切片在光镜下随机选取5个视野,图像输入自动图像分析仪,测量心肌纤维化面积与微动脉内、外径比值及阳性细胞数所占比例。PCNA阳性表达率参照Quaini等5判定方法,按每张切片中阳性心肌细胞数占全部心肌细胞数的百分比,分成级:25为级;