TNF-α及TNF-α抗体对破骨细胞V-ATP酶的影响

目的研究不同浓度的TNF-α及TNF-α抗体对破骨细胞上V-ATP酶表达量的影响。方法体外诱导小鼠RAW264.7细胞分化为破骨细胞,通过抗酒石酸酸性磷酸酶染色检测破骨细胞生成情况。然后将破骨细胞分为对照组、TNF-α干预组及TNF-α抗体干预组,TNF-α干预组、TNF-α抗体干预组分别用低、中、高三种浓度的TNF-α、TNF-α抗体干预48 h。用实时荧光定量聚合酶链反应(real-time PCR)、Western blot检测破骨细胞V-ATP酶的mRNA和蛋白表达水平。结果 TRAP染色检测提示有多核破骨细胞生成。TNF-α处理组V-ATP酶mRNA表达水平显著高于对照组(P0.001);TNF-α抗体处理组V-ATP酶mRNA表达水平显著低于对照组(P0.001)。同时,TNF-α处理组V-ATP酶蛋白表达水平显著高于对照组(P0.05);TNF-α抗体处理组V-ATP酶蛋白表达水平显著低于对照组(P0.05)。结论 TNF-α可提高破骨细胞V-ATP酶的表达;TNF-α抗体可抑制破骨细胞V-ATP酶的表达。上述提示TNF-α可能通过提高破骨细胞V-ATP酶的表达从而增加破骨细胞的骨吸收作用。

Effect of TNF-alpha and TNF-alpha antibody on the V-ATPase expression in osteoclasts

Objective To explore the effect of different concentrations of TNF-alpha and TNF-alpha antibody on the V-ATPase expression in osteoclasts. Methods Mouse RAW264.7 cells were induced into osteoclasts in vitro. The osteoclastogenesis was detected with tartrate resistant acid phosphatase (TRAP) staining. The osteoclasts were divided into control group, TNF-alpha intervention group, and TNF-alpha antibody intervention group. TNF-alpha antibody intervention group was treated with different concentrations of TNF-alpha antibody for 48h. The mRNA and the protein expression levels of V-ATPase in osteoclasts were examined using real-time PCR and Western blotting analysis. Results The formation multinucleated osteoclasts were proved with TRAP staining. The mRNA expression level of V-ATPase in TNF-alpha treatment group were significantly higher than that in the control group (P<0.001). The mRNA expression level of V-ATPase in TNF-alpha antibody treatment group were significantly lower than that in the control group (P<0.001). The protein expression levels of V-ATPase in TNF - alpha treatment group were significantly higher than that in the control group (P<0.05). The protein expression level of V-ATPase in TNF-alpha antibody treatment group were significantly lower than that in the control group (P<0.05). Conclusion TNF-alpha improves the expression of V-ATPase in osteoclasts. TNF-alpha antibody reduces the expression of V-ATPase in osteoclasts. These suggest that TNF-alpha improves osteoclastic bone absorption by increase the expression of V-ATPase.