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目的:探讨人多发性骨髓瘤RPMI 8226细胞条件培养液对破骨前体细胞RAW264.7的影响。方法:Western blotting检测可溶性核因子κB受体激活剂配体(soluble receptor activator of NF-κB ligand,sRANKL)的蛋白表达。抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase,TRAP)染色观察RAW264.7细胞的分化成熟情况。RT-PCR法检测RAW264.7细胞TRAP和cathepsin K mRNA的表达。结果:Western blotting法证实RPMI8226细胞条件培养液中含有sRANKL。TRAP染色发现RPMI 8226细胞条件培养液能诱导RAW264.7细胞分化为TRAP阳性的多核成熟破骨细胞。人抑制性RANKL单克隆抗体拮抗30%RPMI 8226细胞条件培养液中sRANKL的作用,且具有剂量依赖性。30%RPMI 8226细胞条件培养液能刺激RAW264.7细胞上调TRAP和cathepsin KmRNA表达。结论:人多发性骨髓瘤细胞RPMI 8226条件培养液中的sRANKL具有促RAW264.7破骨前体细胞分化成TRAP阳性的多核成熟破骨细胞的生物活性。人抑制性RANKL单克隆抗体阻断30%RPMI 8226细胞条件培养液中sRANKL的诱导分化作用,且具有浓度依赖性。
Objective: To investigate the effect of multiple myeloma RPMI 8226 cells conditioned medium on osteoclast precursor RAW264.7. Methods: The protein expression of soluble receptor activator of NF-κB ligand (sRANKL) was detected by Western blotting. Tartrate-resistant acid phosphatase (TRAP) staining was used to observe the differentiation and maturation of RAW264.7 cells. The mRNA expression of TRAP and cathepsin K in RAW264.7 cells was detected by RT-PCR. Results: Western blotting confirmed that sRANKL was contained in RPMI8226 cell conditioned medium. TRAP staining found that RPMI 8226 cell conditioned medium can induce RAW264.7 cells to differentiate into TRAP-positive multinucleated mature osteoclasts. Human inhibitory RANKL monoclonal antibodies antagonized the effect of sRANKL in 30% RPMI 8226 cell conditioned medium in a dose-dependent manner. 30% RPMI 8226 cells conditioned medium can stimulate RAW264.7 cells upregulation of TRAP and cathepsin K mRNA expression. CONCLUSIONS: sRANKL in RPMI 8226 conditioned medium from human multiple myeloma cells has the biological activity of promoting the differentiation of RAW264.7 osteoclast precursors into TRAP-positive mature osteoclasts. Human inhibitory RANKL monoclonal antibody blocks the induction and differentiation of sRANKL in 30% RPMI 8226 cell conditioned medium in a concentration-dependent manner.