中国骨质疏松杂志
中國骨質疏鬆雜誌
중국골질소송잡지
Chinese Journal of Osteoporosis
2015年
10期
1165-1168,1173
,共5页
遆云帆%徐海栋%王瑞%赵建宁
遆雲帆%徐海棟%王瑞%趙建寧
제운범%서해동%왕서%조건저
破骨细胞%绿色荧光蛋白%活细胞成像%细胞骨架%微管正端蛋白
破骨細胞%綠色熒光蛋白%活細胞成像%細胞骨架%微管正耑蛋白
파골세포%록색형광단백%활세포성상%세포골가%미관정단단백
Osteoclast%GFP%Live cell station%Cytoskeleton%EB1
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。
目的:採用活細胞成像技術觀察破骨細胞微管正耑蛋白EB1在細胞內的分佈及運動,初步研究微管在破骨細胞功能中的作用。方法①用脂質體轉染方法(暘離子脂質體Lip2000)轉染EB1?GFP基因至Raw264?7細胞繫,G418篩選轉染成功的Raw264?7細胞,熒光顯微鏡下觀察後GFP蛋白免疫熒光染色確定穩定轉染EB1?GFP的Raw264?7細胞繫建立;②活細胞工作站下觀察轉染EB1?GFP的Raw264?7細胞中EB1蛋白的運動;③用含100ng/mLRANKL和30ng/mL M?CSF的培養基分彆誘導穩定轉染EB1?GFP的Raw264?7細胞與正常Raw264?7細胞為破骨細胞,進行TRAP染色鑒定,比較兩組細胞形態有無差彆;④Raw264?7細胞繫誘導齣破骨細胞後,用細胞免疫熒光染色方法觀察破骨細胞EB1蛋白的形態及分佈;⑤活細胞工作站下觀察穩轉EB1?GFP的Raw264?7細胞誘導齣的破骨細胞內EB1蛋白的運動狀態。結果①脂質體轉染方法建立瞭穩定轉染EB1?GFP基因的Raw264?7細胞繫;②觀察到破骨前體細胞Raw264?7的微管正耑蛋白( EB1)的運動軌跡;③轉染EB1?GFP基因的Raw264?7細胞與正常Raw264?7細胞誘導的破骨細胞TRAP染色無明顯差彆;④活細胞工作站觀察破骨細胞微管正耑蛋白EB1的運動狀態,結果錶明破骨細胞微管活動性較破骨前體細胞Raw264?7活動性低。結論①EB1?GFP基因對破骨前體細胞繫Raw264?7誘導破骨細胞無明顯影響;②微管活動性降低可能與破骨細胞骨吸收活性相關。
목적:채용활세포성상기술관찰파골세포미관정단단백EB1재세포내적분포급운동,초보연구미관재파골세포공능중적작용。방법①용지질체전염방법(양리자지질체Lip2000)전염EB1?GFP기인지Raw264?7세포계,G418사선전염성공적Raw264?7세포,형광현미경하관찰후GFP단백면역형광염색학정은정전염EB1?GFP적Raw264?7세포계건립;②활세포공작참하관찰전염EB1?GFP적Raw264?7세포중EB1단백적운동;③용함100ng/mLRANKL화30ng/mL M?CSF적배양기분별유도은정전염EB1?GFP적Raw264?7세포여정상Raw264?7세포위파골세포,진행TRAP염색감정,비교량조세포형태유무차별;④Raw264?7세포계유도출파골세포후,용세포면역형광염색방법관찰파골세포EB1단백적형태급분포;⑤활세포공작참하관찰은전EB1?GFP적Raw264?7세포유도출적파골세포내EB1단백적운동상태。결과①지질체전염방법건립료은정전염EB1?GFP기인적Raw264?7세포계;②관찰도파골전체세포Raw264?7적미관정단단백( EB1)적운동궤적;③전염EB1?GFP기인적Raw264?7세포여정상Raw264?7세포유도적파골세포TRAP염색무명현차별;④활세포공작참관찰파골세포미관정단단백EB1적운동상태,결과표명파골세포미관활동성교파골전체세포Raw264?7활동성저。결론①EB1?GFP기인대파골전체세포계Raw264?7유도파골세포무명현영향;②미관활동성강저가능여파골세포골흡수활성상관。
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.