培养基对小鼠单核巨噬细胞RAW264.7诱导分化破骨细胞的影响

Effects of different media on osteoclast differentiation induced by RAW264.7 cells

目的 探讨培养基对小鼠单核巨噬细胞RAW264.7向破骨细胞分化的影响。方法 实验分为3组:高糖DMEM培养基组(DMEM组);高糖DMEM/α-MEM培养基组(DMEM/α-MEM组);α-MEM培养基组(α-MEM组)。按常规方法采用上述3种培养基进行破骨细胞培养,培养5 d后,采用抗酒石酸酸性磷酸酶(tartrate resistant acid phosphatase, TRAP)染色观察各组破骨细胞的形成情况,并采用实时荧光定量PCR方法观察破骨细胞分化相关标志物NFATc-1、c-Fos和TRAF-6 mRNA的表达;培养11 d后采用甲苯胺蓝染色进行骨陷窝面积分析,观察破骨细胞骨吸收功能情况。结果 3组均可以观察到典型的TRAP^(+)破骨细胞。与DMEM组相比,DMEM/α-MEM组、α-MEM组TRAP^(+)破骨细胞数量明显增加(P<0.01),但各组形态略有不同。在骨吸收功能上,与DMEM组和DMEM/α-MEM组相比,α-MEM组骨陷窝面积明显增加(P<0.01)。在破骨细胞分化相关调控因子表达上,与DMEM组相比,α-MEM组NFATc-1、c-Fos和TRAF-6 mRNA表达显著增加(P<0.01,P<0.05);DMEM/α-MEM组NFATc-1 mRNA表达显著增加(P<0.01),c-Fos和TRAF-6 mRNA表达有增加的趋势,但差异无统计学意义;α-MEM组与DMEM/α-MEM组比较差异无统计学意义。结论 高糖DMEM培养基、高糖DMEM/α-MEM培养基、α-MEM培养基均可用于小鼠单核巨噬细胞RAW264.7诱导分化破骨细胞的实验;从破骨细胞数量、状态及功能来看,α-MEM培养基更适合做为破骨细胞分化培养基。

Objective To investigate the effects of different culture medium on the differentiation of RAW264.7 cells into osteoclasts, in order to optimize the culture conditions of osteoclasts differentiation. Methods The experiment was divided into three groups: high glucose DMEM differentiation medium group(DMEM group), high glucose DMEM/α-MEM differentiation medium group(DMEM/α-MEM group), α-MEM differentiation medium group(α-MEM group). Tartrate-resistant acid phosphatase(TRAP) staining was used to observe the formation of mature osteoclasts in each group. Toluidine blue staining was used to quantitatively analyze the area of bone lacuna to observe the function of osteoclasts. Real time PCR was used to detect the expression of NFATc-1, c-Fos and TRAF-6 mRNA. Results Typical TRAPosteoclasts were observed in all groups. Compared with DMEM group, the number of TRAPosteoclasts in DMEM/α-MEM group and α-MEM group was significantly increased(P< 0.01). However, the morphology of every group are slightly different. Compared with DMEM group, the mRNA expressions of NFATc-1, c-Fos and TRAF-6 in α-MEM group were significantly increased(P<0.01, P<0.05);Compared with the DMEM group, the mRNA expression of NFATc-1 in DMEM/α-ME/M group was significantly increased(P<0.01), and the mRNA expression of c-Fos and TRAF-6 had a tendency to increase, but there was no statistical difference, there was no significant different between α-MEM group and DMEM/α-MEM group. Conclusion High glucose DMEM differentiation medium, high glucose DMEM/α-MEM differentiation medium and α-MEM differentiation medium can induce RAW264.7 cells to differentiate into osteoclasts, but from the number, morphology and function of osteoclasts, α-MEM medium is more suitable for osteoclast differentiation.