腺病毒介导hIGF-1基因转染对软骨细胞增殖的影响

Effect of transfecting adenovirus-mediated human insulin-like growth factor-1 gene on proliferation of chondrocyte

目的:探讨腺病毒介导人胰岛素样生长因子(human insulin-like growth factor,hIGF-1)基因转染对软骨细胞增殖的影响。方法:构建携带hIGF-1基因的重组腺病毒并进行PCR、Western blot鉴定。体外培养人胚胎软骨细胞,用处于对数增长期的第3代软骨细胞进行实验。分别转染1、10、100及500不同感染复数单位(multiplicity of infection,MOI)的Ad／hIGF-1,用PBS做阴性对照,hIGF-1生长因子(100μg／L)做阳性对照,采用四氮甲基唑蓝(methyl thiazolyl tetrazolium,MTT)法检测不同时间、不同组别软骨细胞吸光度。结果:第2代重组腺病毒上清液中PCR鉴定含有hIGF-1基因,Western blot证实Ad／hIGF-1表达成熟的hIGF-1生长因子。不同病毒滴度转染对软骨细胞增殖的影响存在量效依赖关系,100 MOI软骨细胞吸光度约为对照组3倍,1 MOI与10MOI、500MOI对软骨细胞增殖的影响近似;PBS组随着细胞体外培养时间延长,细胞增殖下降,hIGF-1生长因子、hIGF-1基因对软骨细胞增殖的影响存在时效关系,72 h达到峰值。结论:Ad／hIGF-1基因转染对软骨细胞增殖的影响存在量效、时效依赖关系。

Objective: To explore the effect of transfecting adenovirus-mediated human insulin-like growth factor-1 gene on proliferation of chondrocyte. Methods: Replication defective recombinant adenovirus carrying hIGF-1 gene was constructed and identified with PCR and western blot technique. Human embryo chondrocyte was cultured and the third generation chondrocyte was adopted in this experiments. Chondrocytes were transfected with 1,10,100,500 MOI Ad/hIGF-1, while PBS was taken as negative control, hIGF-1 growth factor (100 μg/L)as positive control. Optical density of the different groups was measured by MTT method at the different stages after transfecting. Results: hIGF-1 gene presented at the second recombinant adenovirus, Ad/hIGF-1 can express hIGF-1 growth factor. The effect of Ad/hIGF-1 transfection on proliferation of chondrocyte displayed the dose dependent relationship that reached the peak at the point of 100 MOI. Optical density of chondrocyte at the 100 MOI was about three times of PBS group, and was similar among the point 1,10 and 500 MOI. Proliferative ability of chondrocyte decreased following the culture time in PBS group. The cell proliferation of Ad/hIGF-1 transfection groups and positive control group existed time dependent relationship that reached the peak at the time of 72 hours after transfecting. Conclusions: Ad/hIGF-1 transfection can stimulate the proliferation of chondrocyte.