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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(multiplicityofinfection,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(methylthiazolyltetrazolium,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,100MOI软骨细胞吸光度约为PBS组的3倍;500MOIAd/CMV-hIGF-1时,软骨细胞吸光度较100MOI下降,与1MOI与10MOI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,?
BACKGROUND: All kinds of growth factors have different promoting effects on chondrocytes cultured in vitro, but their half-lives are short and it is difficult to reach the required aging for the treatment of cartilage defect. Objective: To investigate the effect of self-constructed Ad-CMV-hIGF-1 on chondrocyte proliferation. Design: Set up a controlled experimental study. Location and Subjects: The experiment was conducted at the Lin Baxin Medical Research Center of Sun Yat-sen University. Human embryonic chondrocytes were cultured in vitro, and the third generation of chondrocytes in logarithmic growth phase were used for experiments. Intervention: self-constructed recombinant adenovirus carrying hIGF-1 gene and PCR, Westernblot identification. Generation 3 chondrocytes were transfected with 1, 10, 100 and 500 different multiplicity of infection (MOI) Ad / CMV-hIGF-1, respectively, and treated with phosphate PBS (negative control, hIGF- L) were used as positive control.MAIN OUTCOME MEASURES: The absorbance of chondrocytes at different time and different groups was detected by methylthiazolyltetrazolium (MTT) method.Results: The recombinant adenovirus supernatant of the second generation was used to identify the hIGF -1 gene and Western Blot to confirm the mature hIGF-1 growth factor expressed by Ad / CMV-hIGF-1.There was a dose-dependent relationship between the effects of different virus titer transfection on chondrocyte proliferation, between 1 ~ 100MOI, the chondrocyte absorbance With the increase of virus titer, the absorbance of 100MOI chondrocytes was about 3 times higher than that of PBS group. The absorbance of chondrocytes was lower than that of 100MOI at 500MOIAd / CMV-hIGF-1, and similar to the effect of 1MOI and 10MOI on chondrocyte proliferation. PBS group with cell culture in vitro prolonged?