腺病毒介导的hIGF-Ⅰ基因转染对构建组织工程软骨的影响

Effect of gene transfer of Ad/hrGFP-hIGF-Ⅰon construction of tissue-engineered cartilage

目的探讨腺病毒介导的人胰岛素样生长因子Ⅰ(hIGF-Ⅰ)基因转染对构建组织工程软骨的影响。方法分离新西兰大白兔关节软骨细胞,培养到第一代,用荧光标记的Ad/hrGFP-hIGF-Ⅰ转染,然后接种在PLGA支架上(转染组);未转染的软骨细胞接种在PLGA支架上(对照组),在体外培养2周。利用荧光显微镜、Western blot、RT-PCR、免疫组织化学等方法鉴定hIGF-Ⅰ基因的有效转染,并观察其对构建的组织工程软骨细胞外基质的影响。结果Ad/hrGFP-hIGF-Ⅰ能对兔关节软骨细胞进行有效转染,RT-PCR显示构建的组织工程软骨Ⅰ、Ⅱ型胶原、聚合素的表达高于对照组;定量检测显示胶原和GAG的含量高于对照组(P<0.01)。结论腺病毒介导的hIGF-Ⅰ基因转染能成功转染兔关节软骨细胞,用它构建组织工程软骨能显著促进软骨细胞外基质的合成。

Objective To observe the effects of gene transfer of Ad/hrGFP-hIGF- Ⅰ on the construction of tissue-engineered cartilage. Methods The isolated New Zealand white rabbit articular chondrocytes were cultured to the first generation before transfeetion. Then some of them received IGF-Ⅰ gene transduction by Ad/hrGFP-hIGF-Ⅰ and were seeded into polymer lactic acid-poly-α-lgiutamic acid （PLGA） scaffolds （transfection group）. The others without transfection were taken as control group. After 2 weeks cultivation in vitro, the effective gene transduction and the enhancement of extracellular matrix expression were checked through revert fluorescent microscope, Western blot, RT-PCR and immunohistochemistry. Results Ad/hrGFP-hIGF- Ⅰ could transfect the rabbit articular chondrocytes successfully. Besides, expression of aggrecan, type Ⅱ collagen and type Ⅰ collagen in transfection group were higher than those in control group （P〈0. 01）. Quantitative analysis showed that the significant increases of glycosarninoglycan （GAG） and collagen were more in transfection group than those in control group （P〈0. 01）. Conclusion The IGF- Ⅰ gene transfer of Ad/hrGFP- hIGF- Ⅰ can obviously enhance the construction of tissue-engineered cartilage.