碱性成纤维细胞生长因子转染兔关节软骨细胞的研究(英文)

Transfection of basic fibroblast growth factor in rabbits' articular chondrocytes

背景:软骨细胞体外培养困难和表型难以维持是软骨组织工程研究的一大难题。目的:探讨碱性成纤维细胞生长因子(basicFibroblastGrowthFactor,bFGF)基因转染兔关节软骨细胞后对培养的关节软骨细胞形态、分裂增殖及代谢等方面的影响。设计:完全随机对照实验研究。地点和方法:实验在解放军第一军医大学热带军队卫生学系完成,对象为兔软骨细胞(3周龄新西兰新生兔购于第一军医大学实验动物中心)。干预:将bFGF基因克隆于真核表达载体pHβ0AP-1中,构建重组真核表达载体pHβ-bFGF,转染兔关节软骨细胞。G418筛选阳性克隆,检测阳性细胞bFGF基因的表达水平。测定培养软骨细胞的DNA含量、糖醛酸含量、软骨细胞增殖情况及进行细胞周期分析。主要观察指标:DNA含量、糖醛酸含量、软骨细胞增殖情况及细胞周期分析。结果:bFGF基因转染软骨细胞表型未见显著变化;bFGF基因转染组、载体对照组、空白对照组DNA含量分别为(77.37±6.21),(40.39±4.33),(33.77±4.25)μg/瓶(P<0.01),糖醛酸含量分别为(308.8±10.2),(77.9±8.7),(80.2±10.5)μg/瓶(P<0.01),软骨细胞G1期分别为59.3±2.1,69.5±4.0,73.1±3.9(P<0.05)。结论:bFGF转染关节软骨细胞后,可显著促进细胞分裂增殖并缩短细胞周期。

BACKGROUND:The difficulties in the culture in vitro of chondrocytes and in the maintenance of phenotype are big problems in the research of cartilage engineering. OBJECTIVE:To discuss the impact of basic fibroblast growth factor(bFGF) gene on the morphology,division,proliferation and metabolism of chondrocytes in culture after the transfection in articular chondrocytes of rabbits. DESIGN:A complete randomized controlled study was conducted. SETTING and PARTICIPANTS:The study was completed in the Department of Military Tropical Medicine and Hygiene,First Military Medical University.The subjects were rabbit chondrocytes(New Zealand 3 week old newborn rabbit obtained from Center for Experimental Animals,First Military Medical University). INTERVENTIONS:bFGF gene was cloned into eukaryon expression carrier pHβoAP 1 to construct recombinant eukaryon expression carrier pHβ bFGF for rabbit articular chondrocyte transfection.The positive clones were screened by G418 and the expressive level of bFGF gene in positive cells was detected.The DNA contents,glycuronate contents,and the situation of chondrocyte proliferation were detected as well as the cell cycles were analyzed. MAIN OUTCOME MEASURES:DNA content,glycuronate content,the situation of chondrocyte proliferations,and the analysis of cell cycles. RESULTS:There was no significant change in the phenotype of chondrocytes transfected by bFGF gene.The DNA contents of bFGF gene transfection group,carrier control group,and blank control group were (77.37±6.21),(40.39±4.33) and (33.77±4.25) μg/bottle respectively(P< 0.01); the glycuronate contents were (308.8±10.2),(77.9±8.7) and (80.2±10.5) μg/bottle respectively(P< 0.01);and the G1 phase of chondrocyte were (59.3±2.1),(69.5±4.0) and (73.1±3.9) respectively(P< 0.05). CONCLUSION:The cellular division and proliferation were significantly promoted and the cell cycles were significantly shortened by bFGF transfection in articular chondrocytes,which provide a new technique and theoretical foundation for the researches of cartilage engineering.