人胰岛素样生长因子-1基因转染对软骨细胞增殖的影响

背景各种生长因子对体外培养的软骨细胞均有不同程度的促增值作用,但半衰期较短,很难达到软骨缺陷治疗所要求的时效.目的探讨自行构建携带人胰岛素样生长因子腺病毒载体(Ad/CMV-hIGF-1)对软骨细胞增殖的影响.设计设立对照的实验研究.地点和对象实验在中山大学林百欣医学研究中心进行.体外培养人胚胎软骨细胞,采用对数增长期的第3代软骨细胞进行实验.干预自行构建携带hIGF-1基因的重组腺病毒并进行PCR,Westernblot 鉴定.采用1,10,100及500不同感染复数单位(multiplicity of infection,MOI)的Ad/CMV-hIGF-1分别转染第3代软骨细胞,用磷酸盐(PBS)做阴性对照,hIGF-1生长因子(100μg/L)做阳性对照.主要观察指标采用四氮甲基唑蓝(methylthiazolyl 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,Westernblot证实Ad/CMV-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,在1～100MOI之间,软骨细胞吸光度随着病毒滴度增加逐渐升高,100 MOI软骨细胞吸光度约为PBS组的3倍;500MOI Ad/CMV-hIGF-1时,软骨细胞吸光度较100 MOI下降,与1 MOI与10 MOI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,72h达到峰值.结论成功构建Ad/CMV-hIGF-1;Ad/CMV-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.

Effect of humaninsulin-like growth factor-1 gene transfection on chondrocyte proliferation

BACKGROUND: All the growth factors have the effect of promoting proliferation on the cultured carilage cell in vitro, but it is difficult to maintain the therapeutic effect within sufficient time due to their short half life.OBJECTIVE: To construct a recombinant adenoviral vector carrying the gene encoding human insulin-like growth factor-1(Ad/CMV-hIGF-l), and evaluate its effect on chondrocyte proliferation.DESIGN: A controlled experimental trial was conducted.SETTING and PARTICIPANTS: All experiments were done at the Lin Bai-xin Medical Center of Sun Yet-sen University. Human articular chondrocytes were isolated and cultured in vitro and the third passage of exponential growth were used.INTERVENTIONS: Ad/CMV-hIGF-1 was constructed and identified by PCR and Western blotting. The chondrocytes were infected with Ad/CMV-hIGF-1 for 1, 10, 100, and 500 multiplicity ofinfection(MOI), and PBS was used as the negative control, with hIGF-1 (100 μg/L)as positive the control.MAIN OUTCOME MEASURES: Chondrocyte absorbance in the different groups was measured with MTT assay at different phases after the infection.RESULTS: hIGF-1 gene was identified by PCR in the culture supernatant of the second passage of the recombination adenovirus, and Western blotting verified the expression of mature hIGF-1 by Ad/CMV-hIGF-1. Ad/CMV-hIGF-1 promoted chondrocyte proliferation in a does-dependent fashion, achieving the maximal effect at 100 MOI. Chondrocyte absorbance increased gradually with the titer of the adenovirus applied, and at the 100MOI, it was three times as high as that of the PBS group, and 500 MOI resulted in a lowered absorbance which was similar to that by 1 and 10 MOI of the adenovirus. Chondrocyte proliferation decreased with the prolongation of the culture time in the PBS group. In the Ad/CMV-hIGF-1 group, Ad/CMV-hIGF-1 infection promoted cbondrocyte proliferation also in a time-dependent manner, roaching the peak level 72 hours after the infection, a result similar to that of hIGF-1 treatment.CONCLUSION: Ad/CMV-hIGF-1 has been successfully constructed,which significantly stimulates chondrocyte proliferation in a dose- and time-dependent fashion.