中华口腔医学杂志
中華口腔醫學雜誌
중화구강의학잡지
Chinese Journal of Stomatology
2015年
4期
235-239
,共5页
破骨细胞%伪足%微丝肌动蛋白质类%绿色荧光蛋白质类
破骨細胞%偽足%微絲肌動蛋白質類%綠色熒光蛋白質類
파골세포%위족%미사기동단백질류%록색형광단백질류
Osteoclasts%Pseudopodia%Microfilament proteins%Green fluorescent proteins
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.
目的 觀察增彊型綠色熒光蛋白(enhanced green fluorescent protein,EGFP)標記的Raw264.7體外分化為破骨細胞的能力,擬為EGFP基因作標誌物對外源性破骨前體細胞進行體內追蹤做準備.方法 利用反轉錄病毒介導pEGFP-Lifeact基因轉染Raw264.7細胞;採用有限稀釋技術,熒光顯微鏡下穫得EGFP穩定轉染的G3單剋隆細胞併觀察其形態,將穩定轉染成功的細胞定為G3-EGFP轉染組,未轉染野生細胞作為對照組;覈因子κB受體激活子配體(receptor activator of nuclear factor kappaB ligand,RANKL)誘導G3細胞嚮破骨細胞分化,通過抗酒石痠痠性燐痠酶(tartrate resistant acid phosphatase,TRAP)染色、蛋白質印跡法檢測組織蛋白酶K、骨吸收陷窩實驗觀察轉染後對破骨細胞形成和骨吸收功能的影響;激光共聚焦顯微鏡實時拍攝轉染細胞嚮破骨細胞分化過程中偽足小體結構變化的動態影像.結果 Raw264.7細胞內成功轉染EGFP-Lifeact,傳至20代以上仍可穩定錶達EGFP,建立瞭G3-EGFP單剋隆細胞株;轉染細胞無明顯形態學變化,能誘導形成TRAP染色暘性的多覈巨細胞,轉染組細胞融閤率為(35±5)％,對照組細胞融閤率為(39±5)％,二者差異無統計學意義(P＞0.05);免疫印跡實驗中對照組與轉染組中組織蛋白酶K/β-肌動蛋白半定量分析比值分彆為0.83±0.07、1.02±0.08,兩組細胞組織蛋白酶K錶達差異無統計學意義(P＞0.05);對照組與轉染組骨吸收總麵積分彆為272 252±36 193和262 408±23 243(P＞0.05),骨吸收陷窩數目分彆為320±51和339±55 (P＞0.05),錶明轉染不影響破骨細胞形成與骨吸收功能;激光共聚焦顯微鏡實時拍攝顯示轉染細胞嚮破骨細胞分化時不斷髮生細胞融閤,偽足小體作為動態自組裝結構不斷髮生改建,最初聚集形成偽足小體簇,逐漸形成環狀結構,最終于成熟破骨細胞週圍形成相對穩定的偽足小體帶.結論 EGFP可成功標記Raw264.7細胞;轉染併不影響Raw264.7細胞嚮破骨細胞分化的能力.
목적 관찰증강형록색형광단백(enhanced green fluorescent protein,EGFP)표기적Raw264.7체외분화위파골세포적능력,의위EGFP기인작표지물대외원성파골전체세포진행체내추종주준비.방법 이용반전록병독개도pEGFP-Lifeact기인전염Raw264.7세포;채용유한희석기술,형광현미경하획득EGFP은정전염적G3단극륭세포병관찰기형태,장은정전염성공적세포정위G3-EGFP전염조,미전염야생세포작위대조조;핵인자κB수체격활자배체(receptor activator of nuclear factor kappaB ligand,RANKL)유도G3세포향파골세포분화,통과항주석산산성린산매(tartrate resistant acid phosphatase,TRAP)염색、단백질인적법검측조직단백매K、골흡수함와실험관찰전염후대파골세포형성화골흡수공능적영향;격광공취초현미경실시박섭전염세포향파골세포분화과정중위족소체결구변화적동태영상.결과 Raw264.7세포내성공전염EGFP-Lifeact,전지20대이상잉가은정표체EGFP,건립료G3-EGFP단극륭세포주;전염세포무명현형태학변화,능유도형성TRAP염색양성적다핵거세포,전염조세포융합솔위(35±5)％,대조조세포융합솔위(39±5)％,이자차이무통계학의의(P＞0.05);면역인적실험중대조조여전염조중조직단백매K/β-기동단백반정량분석비치분별위0.83±0.07、1.02±0.08,량조세포조직단백매K표체차이무통계학의의(P＞0.05);대조조여전염조골흡수총면적분별위272 252±36 193화262 408±23 243(P＞0.05),골흡수함와수목분별위320±51화339±55 (P＞0.05),표명전염불영향파골세포형성여골흡수공능;격광공취초현미경실시박섭현시전염세포향파골세포분화시불단발생세포융합,위족소체작위동태자조장결구불단발생개건,최초취집형성위족소체족,축점형성배상결구,최종우성숙파골세포주위형성상대은정적위족소체대.결론 EGFP가성공표기Raw264.7세포;전염병불영향Raw264.7세포향파골세포분화적능력.
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.
