骨髓瘤细胞诱导破骨前体细胞分化的分子机制探讨

Molecular Mechanisms of Human Myeloma Cell Inducing Osteoclastogenesis from Pre-osteoclast Cells

目的探讨人骨髓瘤细胞RPMI8226诱导破骨前体细胞分化的分子机制。方法采用RT-PCR和Western blot-ting法检测RPMI8226细胞能表达核因子κB受体活化因子配基(receptor activator of NF-κB Ligand,RANKL)蛋白和RANKL裂解酶(TACE、ADAM19)mRNA表达。抗酒石酸酸性磷酸酶(TRAP)细胞化学染色鉴定成熟破骨细胞。利用重组人RANKL蛋白(rhRANKL)、条件培养液和人抑制性RANKL单克隆抗体(RANKL mAb)参与培养,诱导RAW264.7细胞分化为成熟破骨细胞。结果 Western blotting法证实RPMI8226细胞表达跨膜型和可溶型RANKL(mRANKL,sRANKL)。RT-PCR法证明RPMI8226细胞表达RANKL、RANKL裂解酶和TRAP mRNA。TRAP染色观察RPMI8226细胞、MG-63细胞条件培养液与rhRANKL均能明显诱导RAW264.7细胞分化为TRAP阳性多核成熟破骨细胞。RT-PCR法证实此3组能刺激RAW264.7细胞上调TRAP mRNA表达。TRAP染色和TRAP mRNA表达中发现RANKL mAb能阻断RPMI8226细胞条件培养液和rhRANKL诱导的破骨前体细胞分化成熟作用。结论骨髓瘤RPMI8226细胞能表达RANKL,其条件培养液可能含sRANKL,能使RAW264.7细胞分化成TRAP阳性多核成熟破骨细胞。

Objective To research the molecular mechanisms of human myeloma cell line RPMI8226 inducing osteoclastogenesis from murine pre - osteoclast monocytie cell line RAW264.7 in vitro. Methods It was studied that RPMI8226 cell could express receptor activator of NF- KB Ligand （RANKL）protein and mRNA , RANKL proteases （TACE,ADAMI9）mRNA by Western blotting and RT- PCR analysis. The morphological change of mature osteoclast was identified by tartrate- resistant acid phosphatase （TRAP）staining. RAW264.7 cell was induced to differentiate to mature osteoclast by culturing with recombinant human RANKL protein （rhRANKL） ,con- ditioned medium , monoclonal anti - human RANKL antibody for neutralization of bioactivity （ RANKL mAb）. Results The protein of membrane and souble RANKL（ mRANKL, sRANKL）were detected by Western blotting in RPMI8226 cell. The mRNA of RANKL, RANKL proteases and TRAP were determined by RT - PCR analysis. The conditioned medium of RPMI8226,MG -63 cell and rhRANKL could induce RAW264.7 cell to differentiate to TRAP- positive multi -nuclei osteoclast by TRAP staining. The rhRANKL,30% MG -63 and RPMI8226 cell conditioned medium could respectively increase the expression of TRAP mRNA in RAW264.7 cells by RT - PCR. Accord- ing to TRAP staining and TRAP mRNA expression,it was confirmed that RANKL mAb could specifically neutralize the biological effect of rhRANKL and 30% RPMI8226 conditioned medium. Conclusion Human myeloma cell line RPMI8226 expressed RANKL. The condi- tioned medium of RPMI8226 cell may secret sRANKL,which had the bioactivity to induce RAW264.7 cell to differentiate to TRAP - posi- tive multi -nuclei osteoclast.