破骨细胞微管正端蛋白动态成像观察

摘要

目的：采用活细胞成像技术观察破骨细胞微管正端蛋白EB1在细胞内的分布及运动，初步研究微管在破骨细胞功能中的作用。方法①用脂质体转染方法（阳离子脂质体Lip2000）转染EB1⁃GFP基因至Raw264�7细胞系，G418筛选转染成功的Raw264�7细胞，荧光显微镜下观察后GFP蛋白免疫荧光染色确定稳定转染EB1⁃GFP的Raw264�7细胞系建立；②活细胞工作站下观察转染EB1⁃GFP的Raw264�7细胞中EB1蛋白的运动；③用含100ng／mLRANKL和30ng／mL M⁃CSF的培养基分别诱导稳定转染EB1⁃GFP的Raw264�7细胞与正常Raw264�7细胞为破骨细胞，进行TRAP染色鉴定，比较两组细胞形态有无差别；④Raw264�7细胞系诱导出破骨细胞后，用细胞免疫荧光染色方法观察破骨细胞EB1蛋白的形态及分布；⑤活细胞工作站下观察稳转EB1⁃GFP的Raw264�7细胞诱导出的破骨细胞内EB1蛋白的运动状态。结果①脂质体转染方法建立了稳定转染EB1⁃GFP基因的Raw264�7细胞系；②观察到破骨前体细胞Raw264�7的微管正端蛋白（ EB1）的运动轨迹；③转染EB1⁃GFP基因的Raw264�7细胞与正常Raw264�7细胞诱导的破骨细胞TRAP染色无明显差别；④活细胞工作站观察破骨细胞微管正端蛋白EB1的运动状态，结果表明破骨细胞微管活动性较破骨前体细胞Raw264�7活动性低。结论①EB1⁃GFP基因对破骨前体细胞系Raw264�7诱导破骨细胞无明显影响；②微管活动性降低可能与破骨细胞骨吸收活性相关。%Objective Using live cell imaging technique to observe the distribution and movement of positive end of microtubule protein EB1 in osteoclasts, and to preliminarily study the function of microtubules in osteoclasts. Methods ( 1 ) EB1⁃GFP was transfected to Raw264�7 stable cell lines using LipofectamineTM 2000. Successful transfected cells were confirmed using immunofluorescence staining of GFP under a fluorescence microscope. (2) EB1⁃GFP in Raw264�7 cells was observed using a living cell station. ( 3 ) EB1⁃GFP⁃transfected Raw264�7 stable cell line and normal Raw264�7 cells were induced by the medium containing 100 ng/ml RANKL or 30 ng/ml M⁃CSF. TRAP staining was used to identify osteoclasts. Cell morphology was compared between the two groups. ( 4 ) EB1 morphology and distribution in the osteoclast was observed after induction by Raw264�7 cells. (5) The running track of EB1⁃GFP was observed in induced osteoclasts by the stable cell line using a living cell station. Results (1) A stably Raw264�7 cell line expressing EB1⁃GFP was established using LipofectamineTM 2000. (2) The running track of EB1⁃GFP was observed in Raw264�7 cells. ( 3 ) TRAP staining showed that no significant difference between osteoclasts induced by stable cell line and normal cells. ( 4 ) The running track of EB1⁃GFP in osteoclasts was observed using a living cell station. Microtubule dynamics was lower in osteoclasts than in Raw264�7 cells. Conclusion EB1⁃GFP gene has no significant effect on osteoclast induction by osteoclast precursor cell Raw264�7. The decrease of microtubule dynamics may be associated with the resorption activity of osteoclasts.