IL-6对RAW264.7细胞成熟分化的体外实验研究

Effect of interleukin-6 on the differentiation of RAW264.7 cells in vitro

目的探讨研究白介素-6(Interleukin-6,IL-6)对核因子NF-κB受体活化因子配体(Receptor activator of nuclear kappa B ligand,RANKL)及对破骨前体细胞的成熟分化和溶骨效应。方法破骨前体细胞RAW264.7细胞经50ng/mL RANKL诱导1 d后将其分为:1、空白对照组(RANKL+PBS)2、低浓度IL-6组(RANKL+50ng/mL IL-6)3、中浓度IL-6组(RANKL+100ng/mL IL-6)4、高浓度IL-6组(RANKL+150ng/mL IL-6)。连续培养9 d后,进行HE染色检测成熟破骨细胞生成量;通过抗酒石酸酸性磷酸酶(Tartrate resistant acid phosphatase, TRAP)染色法观察TRAP阳性多核细胞的情况;运用扫描电镜检测破骨细胞在骨片上的骨吸收陷窝形成情况。结果 HE染色中,成熟破骨细胞生成量中、高浓度IL-6组明显少于低浓度IL-6组(P<0.05),低浓度IL-6组和空白对照组间无明显差别(P>0.05)。②通过TRAP染色后,经染色阳性区域面积与视野面积的百分比计算,中、高浓度IL-6组与明显少于低浓度和空白对照组(P<0.05)。③扫描电镜观察发现骨吸收陷窝面积与视野面积的百分比随着IL-6浓度的增高,相比空白对照组有显著减少,且高浓度IL-6组中陷窝形成最少(P<0.05)。结论 IL-6能直接作用于经RANKL诱导的RAW264.7细胞,能明显抑制破骨细胞激活分化,并降低破骨细胞所致的骨吸收效应。当IL-6浓度超过50ng/mL时,其抑制破骨细胞的骨吸收效应更加明显。

Objective To investigate the effect of interleukin-6(IL-6),a prototypical cytokine featuring pleiotropic and redundant activity,on the osteoclastic differentiation of RANKL-stimulated RAW264.7 macrophages.Methods RAW264.7 cells were pre-treated with 50ng/mL RANKL for 1 day and followed by IL-6 treatment.Then they were incubated and divided into 4 groups:control group(50ng/mL RANKL+PBS),low(50ng/mL RANKL+50ng/mL IL-6),medium(50ng/mL RANKL+100ng/mL IL-6),and high concentration(50 ng/mL RANKL+150ng/mL IL-6)group for 9 consecutive days.These cells were harvested and stained with hematoxylin-eosin(HE)and tartrate-resistant acid phosphatase(TRAP).The resorption pits of bone slices were observed using scanning electron microscope(SEM).Results HE stains showed that there were increased mature osteoclast cells in the control compared to 100 and 150ng/mL of IL-6 treatment,with a difference being statistically significant(P<0.05).However,the difference did not reach statistical significance between the control and 50ng/mL IL-6 treatment(P >0.05).Similarly,compared to the control,TRAP-positive multinucleated osteoclasts(more than 3 nucleus)significantly decreased under the constant stimuli of 100 and 150ng/mL of IL-6 concentration(P<0.05).Under SEM observation,multiple bone resorption formation was clearly visualized in control group.In contrast,few formations of bone resorption and mature osteoclasts were found when the addition of IL-6 concentration was more than 50ng/mL(P<0.05).Conclusion IL-6 at 100 or 150ng/mL concentrations can powerfully suppress the differentiation of mature osteoclasts,thereby resulting in a remarkably reduction of osteolysis.