摘要
目的 利用慢病毒载体（pVSVG）构建表达双色荧光蛋白的单克隆RAW264.7细胞株及其功能鉴定,以期实时检测它们在破骨细胞形成过程中的表达及动态变化.方法 利用pVSVG构建表达双色荧光蛋白的单克隆RAW264.7细胞株,采用激光扫描共聚焦显微镜活细胞成像技术连续动态观察RAW264.7细胞在NF-κB受体激活蛋白配体诱导下向破骨细胞形成的过程.采用抗酒石酸酸性磷酸酶染色（tartrate-resistant acid phosphatase staining,TRAP）及4＇,6-二脒基-2-苯基吲哚（4’,6-diamidino-2-phenylindole,DAPI）染色对表达磷脂酰丝氨酸（phosphatidylserine,PS）与超正电荷的RAW264.7细胞株及破骨细胞进行功能鉴定.结果 联合转染的RAW264.7细胞株形态为圆形或椭圆形,为正常生长形态,且保留了良好的破骨细胞分化能力.超正电荷绿色荧光蛋白（＋ 8-greenfluorescent protein,＋8-GFP）主要表达在RAW细胞的质膜上,分布均匀;当破骨细胞形成后,＋8-GFP在质膜上的荧光强度明显减弱;PS表达在RAW细胞的质膜上,但在破骨细胞形成后,PS不仅表达在质膜上,同时也分布在细胞质和细胞器上.活细胞成像观察发现破骨细胞融合可发生在贴壁状态下及单核与单核、单核与多核及多核与多核细胞之间,最终形成巨大的多核破骨细胞,因此破骨细胞的融合具有反复性.结论 成功构建可同时表达双色荧光蛋白的单克隆细胞株,为在三维空间进一步研究膜融合过程中的各种蛋白质和其他分子的构象改变,以及它们之间的相互作用方式提供了平台.
Objective To establish the transgenic RAW264.7 monoclone cell line labelled by green fluorescent protein（GFP） and modified red fluorescent protein（mRFP）,for investigating the mechanism of cell membrane fusion and cell biological behavior during osteoclastogenesis.Methods A dual-labeling technique involving GFP and mRFP was applied to RAW264.7 cell line by pVSVG for in situ monitoring of membrane fusion during osteoclastogenesis.The live-cell imaging technology was adopted to consecutively observe the process of osteoclast formation induced by receptor activator of NF-κB ligand（RANKL）.Furthermore,tartrate-resistant acid phosphatase staining（TRAP） and 4＇,6-diamidino-2-phenylindole（DAPI） staining were also used to identify the function and characteristics of RAW264.7 monoclonecell line transfected with phosphatidylserine（PS） and ＋ 8 membrane charge.Results The normal morphology of RAW264.7 monoclonecell line transfected with ＋ 8-GFP and PS-mRFP was preserved.The PS and （8 ＋） biosensors co-expressed on the membrane of monocytes.No significant difference of fluorescence density was found.In osteoclasts,（8 ＋）probes disappeared,while PS expressed in both internal organelles and membrane of osteoclasts.Live-cell imaging observation showed that the multinuclear osteoclasts were generated among monocytes and apocytes.All fusion processes occurred under the condition of cell adherence.Conclusions Successful construction of transgenic RAW264.7 monoclone cell line by GFP and mRFP tags provided a wide field of vision for further investigating the cytoskeleton and organelles of subcellular spatial dimension in osteoclastogenesis.
出处
《中华口腔医学杂志》
CAS
CSCD
北大核心
2016年第9期552-557,共6页
Chinese Journal of Stomatology
基金
国家自然科学基金（81302535）
关键词
破骨细胞
荧光蛋白
超正电荷
磷脂酰丝氨酸
Osteoclast
Fluorescent protein
Positive membrane charge
Phosphatidylserine
