骨髓瘤细胞表达APE1促进THP-1细胞破骨样分化.doc

PAGE 骨髓瘤细胞表达APE1促进THP-1细胞破骨样分化 周立为，杜 佳，张 亮，程 燚，王 东，谢家印 （400042重庆，第三军医大学大坪医院野战外科研究所肿瘤中心） [摘要] 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 \* GB3 ①将构建的APE1 siRNA表达载体导入U266细胞中。 = 2 \* GB3 ②Western blotting法检测U266细胞中APE1及破骨细胞分化因子（RANKL）蛋白表达。 = 3 \* GB3 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 \* GB3 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 \* GB3 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（P0.01）。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（P0.05）；补充PU266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（P0.05）。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。 补充P [关键词] APE1；骨髓瘤骨病；共培养；THP-1；RNA干扰 [中图法分类号] [文献标志码] A Effects of RNA interference inhibiting the APE1 expression in U266 cells on differentiation of THP-1 cells into osteoclast-like cells Zhou Liwei, Du Jia, Zhang Liang, Cheng Yi, Wang Dong, Xie Jiayin (Cancer Center,Institute of Surgery Research and Daping Hospital,Third Military Medical University,Chongqing, 4000042,China) [Abstract] Objective To investigate the effect of RNA interference suppressing apurinic/apyrimidinic endonuclease(APE1) expression in Multiple Myeloma cell line U266 on differentiation of THP-1 cells into osteoclast-like cells,which is in a contactless co-culture system. Methods = 1 \* GB3 ①Constructed APE1 siRNA expression vector Ad5v2 APE1 siRNA was used to transfect the U266 cells. = 2 \* GB3 ②The protein levels of APE1 and RANKL were detected by Western blotting. = 3 \* GB3 ③A co-culture system of THP-1 cells and U266 cells was established with Transwell insert culturedish. = 4 \* GB3 ④The co-culture system were divided into 3 groups:THP-1+U266 group,THP-1+U266APE1 siRNA group and the control group(THP-1 cells).Osteoclast-like cells were identified through tartrate-resistant acid phosphatase (TRAP) staining.The