CAJ | 학술논문

背景:巨噬细胞集落刺激因子(macrophage colony-stlimulating factor,M-CSF)/核因子_kB受体激活物配体(receptor activatorof nuclear kappa B ligand,RANKL)两种细胞因子协同诱导骨髓干细胞形成破骨细胞是一种较新的,可以获取较高纯度和数量破骨细胞的诱导培养法,但尚缺乏统一的培养标准.目的:建立有效的M-CSF/RANKL诱导小鼠骨髓干细胞诱导分化破骨细胞的培养体系.方法:分离小鼠四肢骨获取骨髓干细胞.将小鼠骨髓干细胞在含有M-CSF的α-MEM培养基中培养24 h,调整细胞浓度为10~7,10~8,10~9L~(-1).然后在培养基中同时加入10μg/LM-CSF和不同质量浓度(20,50,100μg/L)RANKL.行抗酒石酸酸性磷酸酶染色观察干细胞向破骨细胞的转变过程及细胞形态和染色情况,并对各组染色阳性的破骨细胞进行计数,比较不同诱导条件对破骨样细胞数量的影响. 结果与结论:培养3 d后可见少量破骨样细胞,胞质内含许多红色抗酒石酸酸性磷酸酶染色阳性颗粒,细胞内可见淡染的双核;培养6 d后可见大量染色阳性破骨样细胞;培养9 d后出现多核巨型染色阳性破骨样细胞,细胞明显增大,细胞核可达到3个以上.在固定细胞接种浓度条件下,RANKL质量浓度为100 μg/L时诱导分化形成的破骨样细胞数量较另两质量浓度增多(P<0.05);在固定RANKL质量浓度条件下,细胞浓度为10~5 L~(-1)时诱导分化形成的破骨样细胞数量较另两细胞接种浓度增多(P<0.05):细胞接种浓度为10~8L~(-1),RANKL质量浓度为100 μg/L时诱导分化形成的破骨样细胞数量高于其他条件组合(P<0.05).说明在M-CSF/RANKL诱导小鼠骨髓干细胞分化培养破骨细胞的体系中,RANKL最佳质量浓度为100 μg/L,最佳细胞接种浓度为10~8L~(-1).
배경:거서세포집락자격인자(macrophage colony-stlimulating factor,M-CSF)/핵인자_kB수체격활물배체(receptor activatorof nuclear kappa B ligand,RANKL)량충세포인자협동유도골수간세포형성파골세포시일충교신적,가이획취교고순도화수량파골세포적유도배양법,단상결핍통일적배양표준.목적:건립유효적M-CSF/RANKL유도소서골수간세포유도분화파골세포적배양체계.방법:분리소서사지골획취골수간세포.장소서골수간세포재함유M-CSF적α-MEM배양기중배양24 h,조정세포농도위10~7,10~8,10~9L~(-1).연후재배양기중동시가입10μg/LM-CSF화불동질량농도(20,50,100μg/L)RANKL.행항주석산산성린산매염색관찰간세포향파골세포적전변과정급세포형태화염색정황,병대각조염색양성적파골세포진행계수,비교불동유도조건대파골양세포수량적영향. 결과여결론:배양3 d후가견소량파골양세포,포질내함허다홍색항주석산산성린산매염색양성과립,세포내가견담염적쌍핵;배양6 d후가견대량염색양성파골양세포;배양9 d후출현다핵거형염색양성파골양세포,세포명현증대,세포핵가체도3개이상.재고정세포접충농도조건하,RANKL질량농도위100 μg/L시유도분화형성적파골양세포수량교령량질량농도증다(P<0.05);재고정RANKL질량농도조건하,세포농도위10~5 L~(-1)시유도분화형성적파골양세포수량교령량세포접충농도증다(P<0.05):세포접충농도위10~8L~(-1),RANKL질량농도위100 μg/L시유도분화형성적파골양세포수량고우기타조건조합(P<0.05).설명재M-CSF/RANKL유도소서골수간세포분화배양파골세포적체계중,RANKL최가질량농도위100 μg/L,최가세포접충농도위10~8L~(-1).
BACKGROUND: Macrophage colony-stimulating factor (M-CSF)/receptor activator of nudeer kappa B ligand (RANKL), two types of cytokines co-induce myeloid stem cells to form osteoclasts, is a kind of new method to harvest ostaoclasts with high purity and quantity, but there is lack of uniform cultivation standard. OBJECTIVE: To construct an effective M-CSF/RANKL induced mice myeloid stem cells inducing osteoclast differentiation cultivation system. METHODS: Myeloid stem cells ware obtained from ICR mice and then cultured for 24 hours in a-minimum essential medium containing M-CSF, at cell density of 10~7/L, 10~8/L, 10~9/L. Then 10 μg/L M-CSF and 20, 50, 100 μg/L RANKL were added into culture medium. Tartaric-resistant acid phosphatase stained was performed to observe the transition process from stem cell to osteoclast, as well as cell morphology and stain situation after culture, and positive stained osteoclasts were counted. We compared the influence of different induction conditions to the quantity of osteoclast. RESULTS AND CONCLUSION: A small quantity of osteoclasts contained many red positive beads in the intracytoplasm were observed at 3 days. There were positive beads with hypochromatic dikeryon in cells. A large amount of positively stained osteoclests were seen after 6-day cultudng, which maintained dikaryon. After 9-day culturing, positively stained colossal multinudear cells occurred, became larger and maintained three nuclei. At certain cell density, 100 μg/L RANKL could induce to form more osteoclasts compared with other 2 concentrations (P < 0.05); at certain RANKL concentration, the osteoclasts formation at cells density of 10~8/L was dramatically greater than other 2 cell densities (P < 0.05); the number of osteoclasts was the most when the concentration of RANKL was 100 μg/L and cell density of 10~8/L (P < 0.05). When osteoclasts are induced by M-CSF/RANKL from mudne myeloid stem cells, the best concentration of RANKL is 100 μg/L and cells density is 10~8/L.