α-MEM培养基和高糖DMEM培养基对RAW264.7细胞向破骨细胞分化的影响

Influence of DMEM high glucose and α-MEM culture media on osteoclastogenesis differentiation of RAW264.7 cells

目的比较α-MEM和高糖DMEM两种培养基对小鼠破骨细胞前体细胞系RAW264.7细胞分化的影响。方法 (1)根据培养基和是否添加核因子κB受体激活蛋白配体(receptor activator for nuclear factor-κB ligand,RANKL)将细胞分为4组:α-MEM培养基组、添加RANKL的α-MEM培养基组、高糖DMEM培养基组、添加RANKL的高糖DMEM培养基组;(2)于培养第3天收集细胞,分别通过q PCR、免疫印迹实验观察分化相关标记物抗酒石酸酸性磷酸酶(tartrate resistant acid phosphatase,TRAP)、活化的T细胞核因子(nuclear factor of activated T-cells 1,NFATc1)、核因子κB受体活化因子(receptor activator for nuclear factor-κB,RANK)和组织蛋白酶(Cathepsin)K的mRNA及蛋白表达水平,并做TRAP染色观察各组成熟破骨细胞的形成情况,探讨添加RANKL后α-MEM培养基和高糖DMEM培养基对RAW264.7细胞向破骨细胞分化的影响。结果 (1)与添加RANKL的高糖DMEM培养基相比,添加RANKL的α-MEM培养基使RAW264.7细胞的分化相关标记物TRAP、NFATc1、RANK及cathepsin K的mRNA表达水平增加,TRAP、NFATc1及cathepsin K的蛋白表达水平增加;(2)在α-MEM培养基或高糖DMEM培养基中添加RANKL均可使RAW264.7细胞分化为成熟破骨细胞,但添加RANKL的α-MEM培养基处理的细胞组中形成的成熟破骨细胞更多。结论添加RANKL的α-MEM培养基有利于RAW264.7细胞向破骨细胞分化。

Objective To observe the effect of two different media, DMEM high glucose and α-MEM, on the osteoclastogenesis of RAW264.7 cells.Methods （1） Experimental groups were defined according to the presence of receptor activator for nuclear factor-κB ligand （RANKL） and culture media： DMEM high glucose group, α-MEM group, DMEM high glucose ＋RANKL group, and α-MEM ＋RANKL group.（2） After culturing for 3 days, the mRNA and protein expression of differentiation-related markers, tartrate resistant acid phosphatase （TRAP）, nuclear factor of activated T-cells 1 （NFATc1）, receptor activator for nuclear factor-κB （RANK） and cathepsin K were detected by qPCR and western blot, respectively.The number of mature osteoclasts were detected by TRAP staining.Results （1） Compared with cells cultured in DMEM high glucose added with RANKL media, the markers related to osteoclastogenesis were upper-regulated when RAW264.7 cells were cultured in α-MEM media with the presence of RANKL, both in mRNA and protein levels.（2） After added with RANKL, RAW264.7 cells could differentiate into mature osteoclasts no matter cultured in α-MEM or DMEM high glucose media, but the change was more prominent in the α-MEM ＋RANKL group.Conclusion The α-MEM added with RANKL culture media is a better media for the characterization of RAW264.7 cells.