猪心脏瓣膜成肌纤维细胞的分离、鉴定与培养  

Isolation, identification and culture of porcine heart valve myofibroblasts

作　　者：刘凤丹[1] 胡伟林[1] 陈正平[1] 李永胜[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院急诊科,湖北省武汉市430000

出　　处：《中国组织工程研究》2016年第51期7684-7689,共6页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金(81070190)~~

摘　　要：背景:瓣膜间质细胞是心脏瓣膜中的主要细胞成分,成肌纤维细胞是一种能够表达平滑肌α-平滑肌肌动蛋白和Ⅰ型胶原纤维,并具有一定分化潜能的重要的瓣膜间质细胞,不仅在瓣膜的结构中起着支架作用,尤其在瓣膜的正常生理以及病理应答过程中发挥着重要的调控作用。目的:建立一套可靠的心脏瓣膜成肌纤维细胞分离、原代培养与鉴定的方法,为心瓣膜钙化等疾病的进一步研究奠定基础。方法:1从猪心中获取主动脉辨,分别用酶联和消化法、普通酶消化法以及组织块种植法提取猪主动脉瓣瓣膜成肌纤维细胞,进行原代培养;2显微镜观察3种方法培养出的成肌纤维细胞的活性和形态;3用光学显微镜和免疫细胞化学方法鉴定细胞。结果与结论:13种方法中,利用胰酶和胶原酶Ⅱ联合消化,然后迅速终止消化的方法获得成肌纤维细胞的活性更高,状态更好,纯度更高;2在荧光显微镜下,观察到获得的主动脉瓣成肌纤维细胞α-平滑肌肌动蛋白染色阳性,抗血管性血友病因子染色阴性,提示培养的细胞为成肌纤维细胞。BACKGROUND：Valvular interstitial cel s are the main components of the heart valves. Myofibroblasts, as a kind of valvular interstitial cel s, can express alpha-smooth muscle actin and type Ⅰ col agen fiber, and hold differentiation potential. These cel s cannot only play a support role in the valve structure, but also play a regulatory role in the process of the valve normal physiological and pathological responses. OBJECTIVE：To obtain a reliable method of separation, primary culture and identification of myofibroblasts laying a foundation for further study on the cardiac valvular calcification. METHODS：Aortic valve myofibroblas extracted from porcine hearts were primary cultured by trypsin and col agenase combined digestive method, common enzyme-digestion method and tissue-culture method, respectively. The myofibroblast activity and morphology were observed using microscope, and myofibroblasts were identified using light microscope and immunocytochemistrial method. RESULTS AND CONCLUSION：Myofibroblasts had a higher activity and purity cultured by trypsin combined with col agenase II digestion method. Aortic valve myofibroblasts were positive for alpha-smooth muscle actin and negative for von Wil ebrand factor under fluorescence microscope, suggesting that myofibroblasts were successful y obtained.

关 键 词：细胞培养技术 细胞分离 肌细胞 平滑肌 组织工程 组织构建 瓣膜钙化 瓣膜间质细胞 成年猪 成肌纤维细胞 平滑肌细胞 细胞培养 分离 纯化 细胞鉴定 国家自然科学基金 

分 类 号：R318[医药卫生—生物医学工程]