绿色荧光蛋白标记的Raw264.7体外诱导分化为破骨细胞的实验研究

Osteoclast differentiation of Raw264.7 cells expressing enhanced green fluorescent protein in vitro

目的 观察增强型绿色荧光蛋白（enhanced green fluorescent protein,EGFP）标记的Raw264.7体外分化为破骨细胞的能力,拟为EGFP基因作标志物对外源性破骨前体细胞进行体内追踪做准备.方法 利用反转录病毒介导pEGFP-Lifeact基因转染Raw264.7细胞;采用有限稀释技术,荧光显微镜下获得EGFP稳定转染的G3单克隆细胞并观察其形态,将稳定转染成功的细胞定为G3-EGFP转染组,未转染野生细胞作为对照组;核因子κB受体激活子配体（receptor activator of nuclear factor kappaB ligand,RANKL）诱导G3细胞向破骨细胞分化,通过抗酒石酸酸性磷酸酶（tartrate resistant acid phosphatase,TRAP）染色、蛋白质印迹法检测组织蛋白酶K、骨吸收陷窝实验观察转染后对破骨细胞形成和骨吸收功能的影响;激光共聚焦显微镜实时拍摄转染细胞向破骨细胞分化过程中伪足小体结构变化的动态影像.结果 Raw264.7细胞内成功转染EGFP-Lifeact,传至20代以上仍可稳定表达EGFP,建立了G3-EGFP单克隆细胞株;转染细胞无明显形态学变化,能诱导形成TRAP染色阳性的多核巨细胞,转染组细胞融合率为（35±5）％,对照组细胞融合率为（39±5）％,二者差异无统计学意义（P＞0.05）;免疫印迹实验中对照组与转染组中组织蛋白酶K/β-肌动蛋白半定量分析比值分别为0.83±0.07、1.02±0.08,两组细胞组织蛋白酶K表达差异无统计学意义（P＞0.05）;对照组与转染组骨吸收总面积分别为272 252±36 193和262 408±23 243（P＞0.05）,骨吸收陷窝数目分别为320±51和339±55 （P＞0.05）,表明转染不影响破骨细胞形成与骨吸收功能;激光共聚焦显微镜实时拍摄显示转染细胞向破骨细胞分化时不断发生细胞融合,伪足小体作为动态自组装结构不断发生改建,最初聚集形成伪足小体簇,逐渐形成环状结构,最终于成熟破骨细胞周围形成相对稳定的伪足小体带.结论 EGFP可成功标记Raw264.7细胞;转染并不影响Raw264.7细胞向破骨细胞分化的能力.

Objective To observe the osteoclast differentiation of Raw264.7 strain stably expressing enhanced green fluorescent protein（EGFP）.Methods Raw264.7 cells were transfected with EGFP-Lifeact gene via retrovirus.The G3 cell clone was obtained by limited dilution technique which stably expressed EGFP under the fluorescence microscope.The morphology of G3 cells were observed.The effects of transfection on receptor activator of nuclear factor kappaB ligand（RANKL）-induced osteoclastogenesis and bone resorbing function of G3 cells were examined by tartrate resistant acid phosphatase（TRAP） staining,immunoblotting detection of cathepsin K and bone pit resorption assay.The real-time images of podosome dynamics were taken by laser confocal microscopy during osteoclast differentiation of G3 cells.Results The Raw264.7 cells were successfully transfected with EGFP-Lifeact gene.The G3-EGFP cloned strain which could stably express EGFP even after 20 passages was constructed.There was no significant difference in morphology between G3-EGFP and wild Raw264.7 cells.The fusion rates of the transfection group and of the wild control group were （35±5）% and （39±5）%,respectively,which were not significantly different（P〉0.05）.The semi-quantitative ratio of cathepsin K/β-actin in the wild control group and in the transfection group was 0.83±0.07 and 1.02±0.08（P〉0.05）,respectively.Bone pit results showed that the total area of the bone resorption was respectively 272 252±36 193 and 262 408±23 243（P〉0.05） and the number of the bone pits was respectively 320 ± 51 and 339± 55（P〉0.05）.The photos of laser confocal microscopy showed the constant cell-cell fusion during osteoclast differentiation of G3-EGFP cells.In addition,the dynamic self-organized podosome initially assembled podosome clusts,then dynamic rings,finally formed the characteristic podosome belt pattern in mature osteoclasts.Conclusions Enhanced green fluorescent protein high effectively expressed in Raw264.7.Biological character does not change after transfection.