摘要
背景:在关节软骨损伤修复的过程中,由于目前实验室中常用的细胞因子存在半衰期短、价格昂贵等缺点,不能广泛应用于临床。目的:假设结缔组织生长因子对体外培养的兔关节软骨细胞增殖和表型有一定的影响,为组织学方法修复关节软骨缺损寻找新的细胞因子。设计、时间及地点:随机区组设计对照实验,于2007-08/2008-02在深圳龙岗中心医院完成。材料:健康1月龄新西兰大白兔2只;结缔组织生长因子为Propetech公司产品。方法:获取兔关节软骨组织,将其剪碎,Ⅱ型胶原酶消化法原代分离培养兔关节软骨细胞,待细胞铺满瓶底80%胰酶消化传代,取其第2代,细胞贴壁后随机分组,实验组分别加入30,50,100,150μg/L的结缔组织生长因子,对照组仅加入DMEM培养液进行体外培养。主要观察指标:采用倒置相差显微镜下观察细胞形态及数量改变,采用四唑盐(MTT)法检测不同浓度的结缔组织生长因子对软骨细胞增殖的影响,免疫组织化学法(SABC法)检测Ⅱ型胶原的表达情况。结果:①结缔组织生长因子作用下的兔关节软骨细胞生长迅速,呈典型的软骨细胞形态,部分细胞聚集成团,形成软骨结节。②结缔组织生长因子能显著促进兔关节软骨细胞增殖,与对照组相比差异具有显著性意义(P<0.01)。不同质量浓度的结缔组织生长因子均能促软骨细胞增殖,100μg/L为最佳作用质量浓度(P<0.01)。③结缔组织生长因子作用下的兔关节软骨细胞Ⅱ型胶原表达增多,且始终为阳性。结论:结缔组织生长因子可以刺激体外培养的兔关节软骨细胞增殖,促进软骨特异性Ⅱ型胶原表达,维持软骨表型。结缔组织生长因子可能用来作为组织工程学修复软骨缺损,治疗骨关节炎的新的生长因子。
BACKGROUND： During the process of articular cartilage recovery, the cytokines commonly used in laboratory are disadvantageous due to shortened half-life and highly cost, which limit their use in clinic. OBJECTIVE： To investigate the effect of connective tissue growth factor （CTGF） on the proliferation and phenotype of rabbit articular chondrocytes cultured in vitro, thus providing a new cytokine for articular cartilage defect recovery in tissue engineering. DESIGN, TIME AND SETTING： The randomized block controlled experiments were performed in Shenzhen Longgang Central Hospital from August 2007 to February 2008. MATERIALS： Two healthy one-month-old New Zealand rabbits were recruited in this study. CTGF was produced by Propetech. METHODS： The harvested rabbit articular cartilage was sheared, and chondrocytes were isolated and cultured by using collagenase Ⅱ digestion. When cells reached 80% confluence, cells were pasaaged by trypsinization. Cells of the second passage were cultured in vitro and randomly divided into two groups after adherence. The experimental groups were cultured in DMEM with 30, 50, 100 and 150 μg/L CTGF, while the control group was given with DMEM only. MAIN OUTCOME MEASURES： Cell morphology and number were observed under an inverted phase contrast microscope. Methyl Thiazolyl Tetrazolium （MTT） assay method was adopted to observe the influence of CTGF with different concentrations to the proliferation of chondrocytes, and immunohistochemical method （SABC） was used to assay the expression of type Ⅱ collagen. RESULTS： ①Rabbit articular chondrocytes under CTGF grew rapidly, which showed a typical chondrocyte appearance, some of them aggregated to form cartilage nodulars. ②CTGF could significantly promote the proliferation of rabbit articular chondrocytes, which had a significant different compared with the control group （P 〈 0.01）. Various concentrations of CTGF were effective to promote the chondrocyte proliferation and the optimal CTGF concentration was 1
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2009年第20期3816-3820,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
