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目的研究在非接触式共培养体系下,RNA干扰多发性骨髓瘤细胞株U266 APE1表达对与其共培养的THP-1细胞破骨样分化的影响。方法①将构建的APE1 siRNA表达载体导入U266细胞中。②Western blot法检测U266细胞中APE1及破骨细胞分化因子(RANKL)蛋白表达。③建立非接触式共培养体系:THP-1+U266共培养组、THP-1+U266APE1 siRNA共培养组和THP-1细胞单培养组。④抗酒石酸酸性磷酸酶(TRAP)染色鉴定破骨样细胞,RT-PCR法检测THP-1细胞Cathepsin K和V-ATPase mRNA表达水平。⑤光镜下观察骨切片陷窝形成。结果 APE1 siRNA能明显抑制U266细胞中APE1及RANKL蛋白表达(P<0.01)。THP-1细胞与U266细胞共培养后,THP-1细胞可分化为TRAP阳性的破骨样细胞,Cathepsin K和V-ATPase基因表达显著升高(P<0.05);U266细胞经APE1 siRNA处理后,共培养体系中THP-1细胞诱导分化的OCLs数量减少,Cathepsin K和V-ATPase基因水平降低,差异均有统计学意义(P<0.05)。结论APE1 siRNA能明显抑制U266细胞诱导的THP-1细胞的破骨样分化,其机制可能与APE1下调U266细胞RANKL有关。
OBJECTIVE: To study the effect of non-contact co-culture system on the osteoclast-like differentiation of THP-1 cells co-cultured with RNAi interferon multi-myeloma cell line U266 APE1. Methods ① The constructed APE1 siRNA expression vector was introduced into U266 cells. Western blot was used to detect the protein expression of APE1 and osteoclast differentiation factor (RANKL) in U266 cells. The establishment of non-contact co-culture system: THP-1 + U266 co-culture group, THP-1 + U266APE1 siRNA co-culture group and THP-1 cell single culture group. (4) TRAP staining to identify osteoclast-like cells. The expression of Cathepsin K and V-ATPase mRNA in THP-1 cells was detected by RT-PCR. ⑤ under light microscope to observe the formation of lacunae. Results APE1 siRNA significantly inhibited APE1 and RANKL protein expression in U266 cells (P <0.01). After THP-1 cells were co-cultured with U266 cells, THP-1 cells could differentiate into TRAP-positive osteoclast-like cells, and the expression of Cathepsin K and V-ATPase genes were significantly increased (P <0.05). After U266 cells were treated with APE1 siRNA The number of OCLs induced by THP-1 cells in co-culture system decreased, while the levels of Cathepsin K and V-ATPase decreased. The difference was statistically significant (P <0.05). Conclusion APE1 siRNA can significantly inhibit the osteoclast-like differentiation of THP-1 cells induced by U266 cells, which may be related to the down-regulation of RANKL in U266 cells by APE1.