摘要
背景:巨噬细胞集落刺激因子(macrophage colony-stlimulating factor,M-CSF)/核因子κB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24h,调整细胞浓度为107,108,109L-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3d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6d后可见大量染色阳性破骨样细胞;培养9d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100μg/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108L-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108L-1,RANKL质量浓度为100μg/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100μg/L,最佳细胞接种浓度为108L-1。
BACKGROUND：Macrophage colony-stimulating factor （M-CSF）/receptor activator of nuclear kappa B ligand （RANKL）,two types of cytokines co-induce myeloid stem cells to form osteoclasts,is a kind of new method to harvest osteoclasts 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 were obtained from ICR mice and then cultured for 24 hours in a-minimum essential medium containing M-CSF,at cell density of 107/L,108/L,109/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 dikaryon in cells. A large amount of positively stained osteoclasts were seen after 6-day culturing,which maintained dikaryon. After 9-day culturing,positively stained colossal multinuclear 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 108/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 108/L （P 0.05）. When osteoclasts are induced by M-CSF/RANKL from murine myeloid stem cells,the best concentration of RANKL is 100 μg/L and cells density is 108/L.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2010年第2期191-195,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
江苏省卫生厅课题资助项目(H200601)~~
