CAJ | 학술논문

核因子κB受体活化因子配体诱导破骨细胞前体的培养与分化
핵인자κB수체활화인자배체유도파골세포전체적배양여분화
Receptor activator of nuclear factor kappa-B ligand induces osteoclast precursor culture and differentiation

万方数据

中国组织工程研究中國組織工程研究 중국조직공정연구
Journal of Clinical Rehabilitative Tissue Engineering Research
2013年 46期 7981-7987 ,共7页

朱伟平%史伟%林琳%李中和%黄瑾

주위평%사위%림림%리중화%황근

组织构建%骨组织构建%核因子κB受体活化因子配体%破骨细胞%细胞培养%凋亡%单核巨噬细胞%矿物质和骨代谢紊乱%肾性骨病%骨髓细胞組織構建%骨組織構建%覈因子κB受體活化因子配體%破骨細胞%細胞培養%凋亡%單覈巨噬細胞%礦物質和骨代謝紊亂%腎性骨病%骨髓細胞
조직구건%골조직구건%핵인자κB수체활화인자배체%파골세포%세포배양%조망%단핵거서세포%광물질화골대사문란%신성골병%골수세포

背景：以往的研究多采用长骨机械分离法获得破骨细胞，破骨细胞为终末分化细胞，无法进一步增殖和传代。因此目前常用骨髓细胞诱导培养法和RAW264.7细胞诱导培养法获得大量的破骨细胞以满足实验需要。 　　目的：探讨细胞核因子κB受体活化因子配体诱导破骨细胞前体细胞分化为成熟破骨细胞的最佳方法。 　　方法：分离小鼠骨髓细胞后添加核因子κB 受体活化因子配体与巨噬细胞集落刺激因子共同诱导或者取RAW264.7细胞单独加入核因子κB受体活化因子配体诱导破骨细胞的形成；分别给予不同浓度的核因子κB受体活化因子配体，观察生成破骨细胞的数量，评价核因子κB受体活化因子配体与破骨细胞生成的量效关系；采用膜联蛋白V-FITC联合PI染色进行流式细胞术分析破骨细胞形成过程中单核巨噬细胞的凋亡情况。 　　结果与结论：当核因子κB受体活化因子配体浓度为10μg/L时，破骨细胞形成数量最多的时间点在第六至七天；而核因子κB受体活化因子配体浓度为100μg/L时，高峰期出现在第四至五天。破骨细胞的形成数量随着核因子κB受体活化因子配体刺激浓度升高而增加，呈浓度依赖性，50μg/L的核因子κB受体活化因子配体是破骨细胞形成数量与浓度关系曲线的转折点，高于150μg/L以后破骨细胞形成数量的增幅明显放缓。核因子κB受体活化因子配体即能诱导单核巨噬细胞形成破骨细胞又可以促进其凋亡，通过破骨细胞计数比较发现在同一浓度(104/cm2)接种单核巨噬细胞后以30μg/L的核因子κB受体活化因子配体诱导后，平均每单位核因子κB受体活化因子配体所获得的破骨细胞数量最多。提示骨髓细胞或RAW264.7细胞诱导破骨细胞的培养方法皆简单可行，细胞接种的最佳浓度为104/cm2；核因子κB受体活化因子配体的适宜刺激浓度为30-50μg/L。
배경：이왕적연구다채용장골궤계분리법획득파골세포，파골세포위종말분화세포，무법진일보증식화전대。인차목전상용골수세포유도배양법화RAW264.7세포유도배양법획득대량적파골세포이만족실험수요。 　　목적：탐토세포핵인자κB수체활화인자배체유도파골세포전체세포분화위성숙파골세포적최가방법。 　　방법：분리소서골수세포후첨가핵인자κB 수체활화인자배체여거서세포집락자격인자공동유도혹자취RAW264.7세포단독가입핵인자κB수체활화인자배체유도파골세포적형성；분별급여불동농도적핵인자κB수체활화인자배체，관찰생성파골세포적수량，평개핵인자κB수체활화인자배체여파골세포생성적량효관계；채용막련단백V-FITC연합PI염색진행류식세포술분석파골세포형성과정중단핵거서세포적조망정황。 　　결과여결론：당핵인자κB수체활화인자배체농도위10μg/L시，파골세포형성수량최다적시간점재제륙지칠천；이핵인자κB수체활화인자배체농도위100μg/L시，고봉기출현재제사지오천。파골세포적형성수량수착핵인자κB수체활화인자배체자격농도승고이증가，정농도의뢰성，50μg/L적핵인자κB수체활화인자배체시파골세포형성수량여농도관계곡선적전절점，고우150μg/L이후파골세포형성수량적증폭명현방완。핵인자κB수체활화인자배체즉능유도단핵거서세포형성파골세포우가이촉진기조망，통과파골세포계수비교발현재동일농도(104/cm2)접충단핵거서세포후이30μg/L적핵인자κB수체활화인자배체유도후，평균매단위핵인자κB수체활화인자배체소획득적파골세포수량최다。제시골수세포혹RAW264.7세포유도파골세포적배양방법개간단가행，세포접충적최가농도위104/cm2；핵인자κB수체활화인자배체적괄의자격농도위30-50μg/L。
BACKGROUND:Previous studies have applied long-bone mechanical separation method to obtain osteoclasts, which are terminal y differentiated cells and cannot further proliferate. Therefore a large number of osteoclasts can be harvested with bone marrow cells inducing culture method and RAW264.7 cells inducing culture method to meet clinical requirements. OBJECTIVE:To investigate the optimal method of receptor activator of nuclear factor kappa-B ligand (RANKL) induced osteoclast precursors to differentiate into mature osteoclasts. METHODS:After bone marrow cells were isolated from mouse, RANKL and macrophage colony stimulating factor were added into the medium together, or RAW264.7 cells were cultured with RANKL to induce osteoclasts. The osteoclast precursors were treated with different concentrations of RANKL to observe the number of generated osteoclasts and evaluate the dose-effect relationship between RANKL and osteoclastogenesis. Annexin V-FITC and propidium iodide staining were used for flow cytometry to analyze the mononuclear-macrophage apoptosis during osteoclastogenesis. RESULTS AND CONCLUSION:When 10μg/L RANKL was used, the peak of osteoclastogenesis appeared at days 6-7;when the concentration of RANKL was up to 100μg/L, the peak appeared at days 4-5. The number of new osteoclasts was dose-dependent on the RANKL concentration. 50μg/L of RANKL was the turning point in the fitted curve from osteoclastogenesis and RANKL concentration. After the RANKL concentration was more than 150μg/L, the number of osteoclasts slowed down obviously. RANKL can induce monocyte-macrophage to form osteoclasts and promote monocyte-macrophage apoptosis. The highest number of osteoclasts would be obtained to each unit of RANKL when monocyte-macrophage cells were cultured with 30μg/L of RANKL in the same vaccination density (104/cm2). Experimental findings indicate that, RAW264.7 cells or bone marrow cells inducing culture methods are simple and feasible, the optimum cellseeding density was 104/cm2;the appropriate stimulation concentration of RANKL was 30-50μg/L.