逆转录病毒载体介导人胰岛素样生长因子-Ⅰ在软骨细胞中的表达

摘要

Objective To transfect the rabbit chondrocytes using a recombinant retroviral vector containing hIGF-I cDNA, and to investigate the expression of exogenous hIGF-I gene in the chondrocytes and its effects on cell biological behavior. Methods The retroviral vector pLNC-IGF-GFP containing targeted gene was transfected into rabbit articular chondrocytes. The positive chondrocytes clones, which were G418-resistant, were selected by G418. The expression of hIGF-I mRNA and protein in chondrocytes were determined by RT-PCR and immunohistochemical staining, respectively. And the expression of GFP gene was observed under fluorescence microscope. Proliferation and phenotype of transfected chondrocytes were tested by MTF, flow cytometry, immunocytochemistry, dimethyl methylene blue method and ELISA. Results The stable expression levels of hIGF-I in transfected chondrocytes were confirmed by RT-PCR and immunocytochemical analysis, and GFP expression was established under fluorescence microscope to be observed green fluorescence. The proliferation of chondrocytes and the secretion of collagen Ⅱ and proteoglycan were higher than those of the same time point in non-transfected chondrocytes within 6 w after transfection ( P ＜0. 01 ). Conclusions 2hIGF-I gene can be effectively transfered into the rabbit chondrocytes mediated by retroviral vector and is expressed stably. Meanwhile, the transfected cells proliferate actively. It may provide a basis for further study of gene therapy for cartilage defects.%目的 探讨逆转录病毒载体介导人胰岛索样生长因子-Ⅰ(human insulin-like growth factor-I,hIGF-I)体外转染兔软骨细胞后软骨细胞hIGF-I的表达情况及其生物学行为变化.方法 将构建的含有目的 基因的逆转录病毒载体pLNc-IGF-GFP体外转染兔关节软骨细胞,经G418筛选阳性克隆,采用RT-PCR及免疫化学染色检测转染软骨细胞中hIGF-I的表达情况,并在荧光显微镜下观察标记基因GFP的表达情况.采用MTT法、流式细胞仪、免疫细胞化学染色、二甲基驱甲蓝法及ELISA法对转染hIGF-I后的软骨细胞的增殖能力及表型进行检测.结果 G418筛选后获得的软骨细胞阳性克隆,经RT-PCR和免疫细胞化学检测表明hIGF-1基因在mRNA和蛋白质水平得到稳定表达,荧光显微镜下观察转染的软骨细胞可激发出绿色荧光,证明转染成功.转染软骨细胞株的增殖能力、Ⅱ型胶原和蛋白多糖的分泌水平,在转染后6 w内均高于同时间点未转染的软骨细胞,差异具有显著性(P<0.05).结论 逆转录病毒载体能有效地将hIGF-I基因转染至兔软骨细胞并获得稳定表达,同时转染后的软骨细胞增生活跃,能够在较长时间维持软骨细胞表型,为进一步研究软骨缺损的组织工程修复和基因治疗打下基础.