Calpain及抑制剂对小鼠破骨细胞分化的作用  

The effect of Calpain and its function of inhibiting murine osteoclastogenesis

作　　者：徐嘉莹[1] 刘薇[1] 黄宇光[1] 

机构地区：[1]中国医学科学院北京协和医学院北京协和医院麻醉科,北京100730

出　　处：《中华骨质疏松和骨矿盐疾病杂志》2011年第2期101-107,共7页Chinese Journal Of Osteoporosis And Bone Mineral Research

摘　　要：目的探讨Calpain及其抑制剂Calpastatin能否抑制核因子一kB活化受体配体(receptor activatorof NFkB ligand,RANKL)诱导的小鼠破骨细胞分化。方法体外培养小鼠RAW264.7细胞,在诱导组分别加入不同浓度的RANKL(0、20、50、70、100 ng/mL),并另设抑制剂组加入RANKL 50 ng/mL和Calpastatin50 nmol/L,共同诱导分化6 d至破骨细胞分化成熟。第2天应用荧光定量分析试剂盒分析各组Calpain活性,并在第6天应用抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase,TRAP)染色和骨凹陷分析方法(pitformation assay)比较各组破骨细胞分化程度的差异。结果 Calpain活性随RANKL剂量的增加而递增(P<0.05),其活性分别与TRAP阳性细胞数和骨凹陷面积呈正相关,相关性具有统计学意义(P<0.05)。Calpastatin抑制剂组与单纯RANKL 50ng/mL诱导组相比,TRAP阳性细胞数和骨凹陷面积均显著降低,差异具有统计学意义(P<0.05)。结论 Calpain是小鼠RAW264.7细胞中RANK L诱导的破骨细胞分化过程中的关键因子,使用Calpastatin抑制Calpain活性可以有效抑制RANKL诱导的破骨细胞分化。Calpain可能成为抑制破骨细胞分化和骨质破坏的新型治疗靶点。Objective To investigate whether Calpain inhibitor Calpaslatin could block RANKL-induced este- oclastogenesis in murine RAW264. 7 cells. Methods Different dosages of RANKL （0 - 100 ng/mL） and RANKL （ 50 ng/mL） plus Calpastatin （ 50 nmol/L） were applied in RAW264. 7 cells respectively for continuous 6 days to induce complete osteoclastogenesis. Calpain activity of each group was measured on day 2 using a fluommetric assay kit. On day 6, TRAP stain and pit formation assay were applied in all groups to determine the maturity of astceclast. Results Cal- pain activity increased in a RANKL dese-dependent manner （ P 〈0. 05 ）. There was a positive correlation between Calpsin activity and TRAP positive cell count, so was it with pit formation area （P 〈0. 05）. Calpain inhibition using C.alpastatin significantly inhibited TRAP positive cell count （ P 〈 0. 05 ） and pit formation area （ P 〈 0. 05 ）, compared with RANKL induction only. Conclusion Calpain is an important factor in RANKL-induced osteeclastogenesis in routine RAW264. 7 cells and its inhibition using Calpastatin can effectively block the esteoclastogenesis process. Therefore, Calpain midst be a novel interveutional target for inhibiting esteoclastogenesis and bone resorption.

关 键 词：CALPAIN 破骨细胞分化 RANKL RAW264.7细胞 骨凹陷 

分 类 号：R681[医药卫生—骨科学]