应用免疫组织化学S-100和α-肌动蛋白抗原鉴定组织工程软骨  

Assessment of tissue-engineered cartilage tissues with soluble protein 100 and alpha-actin

作　　者：纪玲 杨林 武延格 王正 

机构地区：[1]暨南大学第二临床医学院深圳市人民医院检验医学部,广东省深圳市518020 [2]暨南大学第二临床医学院深圳市人民医院胸外科,广东省深圳市518020

出　　处：《中国组织工程研究与临床康复》2008年第28期5554-5557,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金资助项目(30500499)~~

摘　　要：背景:通常采用阿辛蓝染色技术鉴定软骨组织,然而对于组织工程软骨形成早期,阿辛蓝染色不能很好的显示细胞外基质的分泌情况。目的:拟观察采用免疫组织化学法S-100抗原结合α-肌动蛋白抗原染色鉴别人工合成组织工程化软骨结构的可行性。设计、时间及地点:对比观察,于2006-12/2008-02在深圳市人民医院临床医学研究中心完成。材料:2周龄SPF级SD大鼠,体质量50～60g,用于体外培养剑突软骨细胞;DegraPol材料,长20mm,内/外径分别为2.5/4mm,管壁厚度为0.75mm,由瑞士联邦理工大学聚合材料研究所提供。方法:于第3代收集软骨细胞接种于DegraPol管状支架形成软骨细胞-支架复合物,体外培养3周后植入同系大鼠腹腔大网膜体内培养1周。分别于体外培养3周和体内培养1周后取软骨细胞-DegraPol复合物制备组织学检测标本行苏木精-伊红染色和S-100抗原、α-肌动蛋白抗体免疫组织化学染色。主要观察指标:倒置显微镜观察软骨细胞-DegraPol复合物的变化;免疫组织化学染色鉴定软骨细胞。结果:体外培养3周后,苏木精-伊红染色显示软骨细胞位于软骨陷窝内,软骨基质呈蓝色。种植软骨细胞于DegraPol管状泡沫材料支架内侧面,体外培养3周后,在管腔的内侧壁S-100抗原阳性表达,α-肌动蛋白抗原阴性。证实在细胞-支架复合物植入同系大鼠体内培养之前已经形成了新的组织工程化软骨层。置入动物体内培养1周后,所获得的新组织为混合性的细胞结构,S-100抗原阳性多集中在管腔的内侧壁,而α-肌动蛋白抗原阳性反应多集中在管腔的外侧壁。结论:软骨组织S-100抗原阳性表达,结缔组织α-肌动蛋白抗原阳性表达,S-100和α-肌动蛋白抗原可作为组织工程化软骨的鉴定指标。BACKGROUND： Alcian blue staining protocol is usually used to identify cartilage tissues, however, it cannot show the secretion of extracellular matrix well in the early formation stage of tissue-engineered cartilage tissues. OBJECTIVE： To observe the feasibility to identify artificial tissue-engineered cartilage with soluble protein 100 antigen and α -actin antigen immunohistochemical method. DESIGN, TIME AND SETTING： A contrast observation was carried out between December 2006 and February 2008 in the Medical Science Research Center of Shenzhen People＇s Hospital, Shenzhen, Guangdong, China. MATERIALS： Sprague-Dowly rats of SPF grade, aged 2 weeks, weighing 50-60 g, were used to culture xiphoid chondrocytes; DegraPol scaffolds, 20 mm long, 0.75 mm thickness, internal/outer diameter 2.5/4 mm, were provided by Swiss Federal Institute of Technology （Zurich）. METHODS： Passage 3 xiphoid chondrocytes were harvested and seeded onto DegraPol scaffolds to from chondrocyte-polymer composites, and were implanted in the greater omentum of homologous rats to culture in vivo for 1 week following 3-week in vitro static culture. The chondrocyte-polymer composites were harvested after 3-week in vitro static culture and 1-week/n vivo culture, respectively, to perform HE staining and soluble protein 100 and α-actin immunohistochemical staining. MAIN OUTCOME MEASURES： Changes of chondrocyte-polymer composite were observed under inverted microscope; identification of chondrocytes was performed by immunohistocbemistry staining. RUSULTSr After 3-week in vitro static culture, HE staining showed that chondrocytes were in cartilage lacunas and cartilage matrix was stained blue. After implanting chondrocytes and culturing for 3 weeks, the inner side of scaffold was soluble protein 100-positive and a-actin-negative, which indicates chondrocyte-polymer composite has formed new tissue-engineered cartilage layer before implanting in the greater omentum of homologous rats, After l -week in vivo culture, the obtai

关 键 词：软骨细胞 体外培养 组织工程 S-100 Α-肌动蛋白 

分 类 号：R318[医药卫生—生物医学工程]