脂质体介导pIDO-EGFP转染原代培养软骨细胞的初步研究

Preliminary study of primarily cultured C57 articular cartilage transfected with plamid IDO-EGFP by lipofectamine

目的:检测脂质体介导pIDO-EGFP转染原代培养的C57小鼠关节软骨细胞的瞬时表达及转染效率,建立原代培养的小鼠关节软骨细胞转染方法。方法:大肠杆菌中扩增pIDO-EGFP质粒,在最优化条件下通过lipofectamine2000TM转染试剂将pIDO-EGFP质粒转入原代培养的小鼠关节软骨细胞,应用荧光显微镜和激光共聚焦显微镜观察其转染过程及瞬时表达情况,流式细胞术检测其转染效率。结果:质粒携带的增强型绿色荧光蛋白在转染后24h得到了明显表达,48h后流式细胞术检测其转染效率为36.43%,未影响软骨细胞贴壁过程。结论:经绿色荧光蛋白检测表明,脂质体成功地将IDO基因转染进入原代培养的软骨细胞。转染后的软骨细胞在体外仍能存活,在最优化的条件下能达到良好的瞬时转染效率,为组织工程化软骨细胞基因导入和基因修饰提供了思路。

AIM： To determine the transfection efficiency and transient expression of plDO-EGFP gene in primarily cultured C57 articular cartilage of mice, and to establish a transfection method of the primarily cultured articular cartilage in mice. METHODS： Plasmid IDO-EGFP was amplified in Eschen＇chia coli. The primarily cultured mouse chondrocytes which were initially obtained from articular cartilage were cultured in vitro and transfected with plDO-EGFP by lipofectamine2000TM reagent under optimized condition. Transfection process and transient expression were evaluated by fluorescent microscopy and laser scanning confocal microscopy （LSCM）, and transfection efficiency was determined by flow cytometry. RESULTS： There was obvious expression of EGFP at 24 h after transfection. The transfection efficiency of plDO-EGFP into primarily cultured mouse chondrocytes reached 36.43% at 48 hours and the transfection did not affect the process of cell adherence. CONCLUSION： IDO gene has been successfully transfected into primarily cultured chondrocytes by means of lipofectamine 2000TM reagent and the chondrocytes can survive in vitro. Satisfactory efficiency of transient transfection can be reached under optimized condition, which will provide a basis for gene introduction and modification of tissue engineered cartilage.