原代成骨细胞来源胞外囊泡促进破骨细胞分化

Effect of primary osteoblast-derived extracellular vesicles on osteoclast differentiation

目的:观察原代成骨细胞来源胞外囊泡(OB-EV)对破骨细胞增殖、分化的作用,探究胞外囊泡参与成骨细胞和破骨细胞之间通信的可能分子机制。方法:采用酶消化法分离原代成骨细胞,并以β-甘油磷酸钠、抗坏血酸、地塞米松成骨诱导液诱导培养,通过碱性磷酸酶染色及茜素红S染色鉴定成骨细胞分化及矿化功能。收集成骨细胞培养上清液,采用超速离心法提取囊泡分泌物,并通过透射电镜观察囊泡形态,蛋白质印迹法鉴定胞外囊泡表面特征性标志物肿瘤易感基因101(TSG101)、ALG-2相互作用蛋白X(Alix)和分化抗原9(CD9)。采用细胞计数试剂盒8(CCK-8)实验检测OB-EV对RAW264.7的增殖作用。进一步通过蛋白质印迹法检测OB-EV与核因子κB受体活化因子配体(RANKL)联合作用后RAW264.7破骨分化标志物的表达水平。将OB-EV处理的小鼠骨髓来源巨噬细胞(BMM)通过抗酒石酸酸性磷酸酶(TRAP)染色观察并比较破骨细胞数,明确OB-EV对破骨细胞分化的影响。结果:透射电镜观察提取的OB-EV呈直径为30~150 nm的双层杯状结构,且TSG101、Alix、CD9呈阳性表达。CCK-8实验结果显示高浓度(20μg/mL以上)OB-EV抑制RAW264.7增殖(P<0.05)。蛋白质印迹法检测发现RAW264.7中c-Fos、活化T细胞核因子(NFATc1)、c-Jun氨基末端激酶(JNK)等破骨细胞分化标志蛋白表达明显增加,且随OB-EV浓度增加而增加(P<0.05)。此外,将OB-EV与RANKL联合作用于BMM,结果显示TRAP阳性细胞较RANKL对照组显著增多(P<0.05)。结论:OB-EV可促进破骨前体细胞向破骨细胞分化,但高浓度OB-EV会抑制细胞增殖。

Objective:To investigate the effect of osteoblast-derived extracellular vesicles(OB-EVs)on the proliferation and differentiation of osteoclasts,and to explore the possible molecular mechanism of extracellular vesicles involved in the communication between osteoblasts and osteoclasts.Methods:Primary osteoblasts were isolated from newborn mouse calvarial bone and induced byβ-glycero phosphate,ascorbic acid and dexamethasone.Osteogenic feature was tested by alkaline phosphatase(ALP)and alizarin red S staining.Extracellular vesicles were isolated by ultracentrifugation from the cell culture supernatant.Vesicle morphology was observed by transmission electron microscopy,and the characteristic markers of tumor susceptibility gene 101(TSG101),ALG-2 interacting protein X(Alix)and cluster of differentiation 9(CD9)on the surface of extracellular vesicles were identified by Western blotting.Cell counting kit 8(CCK-8)assay was used to determine the proliferation effect of OB-EVs on mouse mononuclear macrophage RAW264.7 cells.Furthermore,the expression level of specific markers of osteoclast differentiation in RAW264.7 cells was detected by Western blotting after the combined effect of OB-EVs and receptor activator for nuclear factorκB ligand(RANKL).The number of osteoclasts was observed and compared with OB-EVs-treated mouse bone marrow-derived macrophages(BMMs)by tartrate-resistant acid phosphatase(TRAP)staining,and the effect of OB-EVs on osteoclast differentiation was determined.Results:The extracted OB-EVs showed a double-layer cup-like structure with a diameter of 30-150 nm,and TSG101,Alix and CD9 were expressed.RAW264.7 cells were stimulated with OB-EVs,and the results of CCK-8 assay showed that high concentration of OB-EVs(more than 20μg/mL)inhibited cell proliferation(P<0.05).Western blotting analysis showed that the expression of osteoclast differentiation marker proteins such as c-Fos,activated T cell nuclear factor(NFATc1)and c-Jun N-terminal kinase(JNK)in RAW264.7 cells were significantly increased,and the promoting effect was enhanced with increasing of OB-EVs concentration(P<0.05).In addition,the combination of OB-EVs and RANKL on BMMs showed that the number of TRAP-positive cells was significantly higher than that of the RANKL induction group alone(P<0.05).Conclusion:OB-EVs can promote the differentiation of osteoclast precursor cells into osteoclasts,but high concentration of OB-EVs can inhibit proliferation of RAW264.7 cells.