摘要
目的:探讨绿色荧光蛋白在组织工程关节软骨体外构建和体内移植修复关节软骨缺损中对细胞的示踪作用。方法:实验于2003-09/12在第三军医大学西南医院中心实验室完成。选取清洁级雄性3～4周龄日本大耳白兔2只作为供体,麻醉处死后,切取关节软骨,系列酶消化分离软骨细胞,洗涤,37℃于体积分数为0.05的CO2中培养,传代扩增。再选取清洁级雄性4月龄日本大耳白兔2只作为受体。首先用反转录病毒载体pLEGFPN1感染软骨细胞,将胶原凝胶包埋的绿色荧光蛋白标记软骨细胞接种于聚磷酸钙纤维/左旋聚乳酸三维支架后体外培养3周,再用标记软骨细胞～支架复合物移植修复兔膝关节软骨缺损。倒置荧光显微镜下观察细胞接种支架率和体外培养过程中细胞在支架上生长、增殖、分化、分布及术后4周细胞在缺损区的成活与软骨缺损修复情况。结果:实验共纳入4只日本大耳白兔,作为受体的2只进入结果分析。①绿色荧光蛋白反转录病毒转染第一代关节软骨细胞情况:转染后3d,倒置荧光显微镜下可看到表达绿色荧光蛋白的关节软骨细胞,主要为三角形,少数呈多角形和梭形。随着传代次数的增加,绿色荧光蛋白阳性软骨细胞的形态变为以梭形为主,少数细胞为三角形和椭圆形,体外培养8周,细胞增殖良好,荧光强度无减弱。流式细胞仪检测G418筛选2周后软骨细胞的绿色荧光蛋白表达率为87.3%。②绿色荧光蛋白标记软骨细胞支架复合物倒置荧光显微镜观察结果:软骨细胞三维接种支架后,细胞几乎全部种植于支架上,在支架材料内分布均匀,刚接种的细胞呈圆形,发出明亮的绿色荧光;3～4h细胞开始变形,2～3d即己基本完全变形,细胞的形态以长梭形为主,呈网状排列,随着培养时间延长,支架上的细胞逐渐由梭形变成圆形,移植前(体外培养3周)支架上细胞大多数己呈圆形。体外培养6�
AIM： To explore the detectable effects of green fluorescent protein （GFP） on the construction of tissue-engineered articular cartilage in vitro and the reparation of articular cartilage defects in vivo. METHODS： The experiment was conducted in the Center Laboratory of Southwest Hospital Affiliated to the Third Military Medical University of Chinese PLA from September to December 2003. Two Japanese flap-eared rabbits at 3-4 weeks of age were selected as donors and killed under anesthesia to obtain articular cartilage so as to extract chondrocytes. After rinse, chondrocytes were cultured in CO2 at a volume fraction of 0.05 at 37℃ for passage and amplification. Another two Japanese flap-eared rabbits at 4 months of age were selected as receptors. Chondrocytes were infected by the recombinant retrovirus pLEGFP-N1 with enhanced GFP. Chondrocytes labeled with green fluorescent protein that were embedded into collagen gel were seeded into three-dimensional calcium polyphophate fibers/poly-L-lactic acid scaffolds. Cell-scaffold complexes were cultured in vitro for three weeks, and then were implanted into articular cartilage defects in established rabbit models. The efficiency of chondrocytes seeding, the growth, proliferation, differentiation and distribution of cells on cell-scaffold complexes cultured in vitro and cell viability in defects four weeks after operation were examined with inverted fluorescence microscope. The repair tissue was assessed histologically with hematoxylineosin and toluidine blue staining. RESULTS： Two rabbits as receptors were involved in the result analysis. ①First-passage chondrocytes： After three days. GFP-labeled chondrocytes almost presented a triangle-shape, and a few presented a polygon-shape or a fusiform-shape. Along with the increased time of passage, GFP-positive chondrocytes were mainly in a shape of a fusiform, and a few in a shape of a triangle or an oval. After 8-week in vitro culture, chondrocytes had a better proliferation and fluorescent intensity was not weak
出处
《中国临床康复》
CSCD
北大核心
2005年第30期84-86,i0003,共4页
Chinese Journal of Clinical Rehabilitation
基金
国家自然科学基金资助项目(30270375)
全军"十五"医药卫生科研重点课题(01Z072)
重庆市重点科技攻关基金资助项目(2003-13-6)~~
