组织工程心肌研究中心肌肌钙蛋白T及细胞间隙连接蛋白43免疫组化检测结果的观察与分析  

Observation and analysis of immunohisto-chemical study of cardiactroponin T and connexin 43 in engineered myocardial tissue studies

作　　者：段翠密[1] 李颖[1] 王海滨[1] 周瑾[1] 刘志强[1] 江红[1] 王常勇[1] 

机构地区：[1]军事医学科学院基础医学研究所组织工程研究室,北京100850

出　　处：《解放军医学杂志》2009年第5期587-590,共4页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金资助项目(30530220);"十一五"863生物技术领域重大项目课题资助项目(2006AA02A105);北京市自然科学基金资助项目(7062053)

摘　　要：目的建立组织工程心肌研究中系统观察免疫组化检测结果的程序与方法。方法取新生大鼠心脏制作石蜡组织切片;用0.25%胰酶消化法分离原代新生大鼠心肌细胞,一部分进行细胞爬片培养,一部分与液态胶原混合构建组织工程心肌并施加力学拉伸刺激,制作组织工程心肌石蜡组织切片。对三者分别进行心肌肌钙蛋白T(cTnT)及细胞间隙连接蛋白43(Cx43)抗体的免疫组化染色。比较观察天然心肌组织、体外培养心肌细胞及组织工程心肌组织免疫组化的检测结果,对其形态结构的变化进行综合分析。分别选取5个不同视野对细胞总数和cTnT阳性细胞进行计数,计算百分比。在细胞和再造心肌组织培养过程中观察自发收缩的细胞区域与cTnT着色区域的关系。结果天然心肌和组织工程心肌切片分别反映了天然在体心肌和体外构建心肌的细胞形态和组织结构特征;心肌细胞爬片显示了体外心肌细胞与非心肌细胞的形态,以及随体外培养时间的延长其形态和数量比例的改变。细胞在体外培养时间越长,成纤维细胞比例越高。尽早将分离的原代新生大鼠心肌细胞与液态胶原混合体外再造心肌组织进行三维拉伸培养有助于抑制其中的成纤维细胞的增殖速度。将分离细胞爬片培养4h后可相继观察到能自发收缩的细胞,其收缩区域与染色片中cTnT的着色区域基本相符;再造的组织工程心肌的收缩自拉伸后48h开始,逐渐从单个细胞到肌束再到整条再造心肌,频率逐渐加快,从1～2/min到5～10/min。结论建立了观察与分析组织工程心肌免疫组化结果的程序和方法,为优化实验设计与条件提供了客观参数,也为构建其他组织的形态学观察与分析方法提供了有利借鉴。Objective To establish the processes and methods of systematical observation and analysis of the results of immunohistochemical and immunocytochemical studies in engineered myocardial tissue studies. Methods The paraffin sections of neonatal myocardial tissue of rat were prepared. The cardiomyocytes of neonatal rats were isolated with 0. 25% trypsinization. Parts of cardiocytes were cultivated on the slides as the sample of cultured cardiocytes, and parts of cardiocytes were mixed with liquid collagen type I to construct the engineered myocardiurn which was then subjected to mechanical stretching. The samples of cultured cardiocytes, paraffin sections of myocardial tissue of neonatal rat and engineered myocardial strands were y stained with cardiactroponin T （cTnT） and connexin43 （Cx43）, respectively, and the results were observed with microscope. The morphological changes in the three groups of cardiocytes were analyzed. The total cellular score and the population of cTnT positive cells were counted with five randomly chosen fields of the samples. The correlation between spontaneous contracting areas and the cTnT positive areas was also observed. Results The samples of natural cardiac tissue and engineered cardiac strand respectively showed the histological structure characters consistent to natural cardiac tissue in vivo and reconstructed cardiac tissue in vitro. While the samples of cultured cardiccytes showed changes in morphological characters, as well as the meristic variation, of cardiac and noneardiac myocytes in vitro as the culture period extended. The proportion of fibroblasts increased as the culture period extended. The addition of liquid collagen type I to cardiocytes isolated from the neonatal rat as early as possible conducing to inhibiting the proliferation of fibroblasts. Spontaneous contraction areas could be observed after cultivation for 4 hours, and the contraction areas matched the cTnT positive areas. 48h after mechanical stretch, the engineered cardiac patch began to contract spon

关 键 词：组织工程 心肌 肌钙蛋白T 连接蛋白43 

分 类 号：R329.29[医药卫生—人体解剖和组织胚胎学]