利用增强型绿色荧光蛋白体外表达探讨逆转录病毒转染软骨细胞的最佳条件

背景:增强型绿色荧光蛋白是目前最佳标记分子,具有荧光特异性高、易于检测等独特的优势,利用基因工程技术构建能稳定表达增强型绿色荧光蛋白的重组逆转录病毒载体EGFP-pLNCX2,转染同种异体软骨细胞值得研究。目的:构建能稳定表达增强型绿色荧光蛋白的重组逆转录病毒载体EGFP-pLNCX2,探讨转染同种异体软骨细胞的最佳条件。设计:随机对照观察。单位:上海交通大学。材料:选用30只出生1周的新西兰白兔,雌雄不限,均购自中国科学院实验动物研究中心。双嗜性逆转录病毒包装细胞系PT67购自Clontech公司;小鼠成纤维细胞NIH3T3细胞购自ATCC公司;DH5a大肠杆菌由本实验室保存;逆转录病毒载体pLNCX2购自Clontech公司;带有增强型绿色荧光蛋白的pEGFP-C1质粒由中科院细胞所丛笑倩教授惠赠。方法:实验于2005-08在上海交通大学完成。分离提取并培养新西兰白兔软骨细胞,利用基因工程技术构建能稳定表达增强型绿色荧光蛋白的重组逆转录病毒载体EGFP-pLNCX2,转染培养后的新西兰白兔软骨细胞,荧光显微镜观察转染的效果。于直径10cm的平皿接种6×105个软骨细胞,分别立即转染及接种12,24,48h后转染逆转录病毒-EGFP,1周后作流式细胞仪测定EGFP表达效率,观察逆转录病毒转染原代软骨细胞最佳时机。软骨酶消化后的软骨细胞接种24h后转染逆转录病毒,分别于第2,3,4,5,6天加250mg/LG418进行筛选。PBS洗涤后作流式细胞仪检测其效率,观察G418筛选的最佳时机。主要观察指标:①EGFP-pLNCX2转染效果。②逆转录病毒转染原代软骨细胞及G418筛选最佳时机。结果:①重组逆转录病毒载体EGFP-pLNCX2转染原代兔软骨细胞,经G418初步筛选而获得的高表达格局,可见软骨细胞经过筛选和表达绿色荧光蛋白后,保持正常的形态学,细胞伸出伪足贴壁,基质分泌旺盛。②逆转录病毒转染原代软骨细胞的最佳时机为细胞接种培养后24h,第7天用流式细胞仪测定转染效率为19.14%,G418筛选的最佳时机为培养后第5天,第7天测定表达效率提升为55.75%。结论:重组逆转录病毒载体EGFP-pLNCX2能有效地转染软骨细胞,逆转录病毒转染原代软骨细胞的最佳时机为细胞接种培养后24h,G418筛选的最佳时机为培养后第5天。

Using in vitro expression of enhanced green fluorescent protein to investigate the optimal conditions for retrovirus transfection into chondrocytes

BACKGROUND: At present,enhanced green fluorescent protein(EGFP)is proved to be the best labeled molecule,with unique advantages,such as high fluorescence specificity,easy to be detected,and so on.Recombined retroviral vector EGFP-pLNCX2,which can stably express EGFP,can be construct using gene-engineering technique.Transfecting allogenic chondrocytes is indeed very useful to investigate the target gene expression and process of constructing tissue engineered cartilage in vivo.OBJECTIVE:To construct recombined retroviral vector EGFP-pLNCX2 which can stably express EGFP,and investigate optimal conditions for retrovirus transfection of chondrocytes.DESIGN:Randomized controlled observation.SETTING:Shanghai Jiao Tong University.MATERIALS:Thirty New Zealand rabbits of either gender,1 week of age,were purchased from Experimental Animal Center of Chinese Academy of Sciences.Amphotropic retrovirus package cell line PT67 pLNCX2 and pEGEP-C1 were purchased from Clontech Company;NIH 3T3 cell line was purchased from ATCC Company;DH5a Bacterium coli was preserved by the laboratory of Shanghai Jiao Tong University;Retroviral vector pLNCX2 was purchased from Clontech Company;pEGFP-C1 plasmid with EGFP were donated by professor Cong Xiao-qian from Chinese Academy of Sciences.METHODS:This experiment was carried out in the Shanghai Jiao Tong University in August 2005.Chondrocytes of New Zealand rabbits were isolated and cultured.The recombinant retroviral vector EGFP-pLNCX2,which can stably express EGFP,was constructed to transfect cultured chondrocytes from New Zealand rabbits by using gene engineering technique.Transfection results were observed under fluorescence microscope.Altogether 6×105 chondrocytes incubated in 10 cm-diameter flat plate were used to transfect retrovirus-EGFP immediately and at 12,24 and 48 hours after inoculation.One week later,EGFP expression efficiency was measured with flow cytometer,and best occasion for retrovirus transfecting primary chondrocytes.Enzyme-digested chondrocytes were inoculated for 24 hours to transfect retrovirus.After 250 mg/L G418 was added,chondrocytes were screened on the 2nd,3rd,4th,5th and 6th days separately.After phosphate buffer solution(PBS)washings,the transfection efficiency of chondrocytes was detected by flow cytometer,and the best occasion for G418 screening was observed.MAIN OUTCOME MEASURES:①EGFP-pLNCX2 transfection efficiency.②The best occasions for retrovirus transfecting primary chondrocytes and G418 screening.RESULTS: ①Retroviral vector EGFP-pLNCX2 transfected primary chondrocytes of rabbits,and high expression of transfected chondrocytes could be obtained through preliminary screening of G418.After being screened and expressing EGFP,chondrocytes kept normal morphology,with pseudopod adhering to the wall and matrix secreting vigorously.②The best occasion for retrovirus transfecting primary chondrocytes was at 24 hours after cell inoculation.The transfection efficiency determined with flow cytometer was 19.14% on the 5th day.The best occasion for G418 screening was on the 5th day after culture.Transfection efficiency of G418 screening was 55.75% on the 7th day.CONCLUSION:Recombinant retroviral vector EGFP-pLNCX2 can effectively transfect chondrocytes.The best occasion for retrovirus transfecting primary chondrocytes is at 24 hours after inoculation, and the best occasion for G418 screening is on the 5th day after culture.