骨髓瘤细胞表达APE1促进THP-1细胞破骨样分化  

APE1 expression in multiple myeloma cells promotes differentiation of THP-1 cells into osteoclast-like cells

作　　者：周立为[1] 杜佳[1] 张亮[1] 程燚[1] 王东[1] 谢家印[1] 

机构地区：[1]第三军医大学大坪医院野战外科研究所肿瘤中心,重庆400042

出　　处：《第三军医大学学报》2013年第16期1663-1666,共4页Journal of Third Military Medical University

基　　金：国家自然科学基金(81172258)~~

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 determine the effect of RNA interference （RNAi） of apurinic/apyrimidinic endonuclease （ APE1 ） in multiple myeloma cell line U266 on the differentiation of a human monocytic cell line THP-1 into osteoclast-like ceils in a contactless co-culture system. Methods Constructed APE1 siRNA expression vector Ad5v2 APE1 siRNA was used to transfect the U266 cells. The protein levels of APE1 and RANKL were detected by Western blotting. A co-culture system of THP-1 cells and U266 cells was established with Transwell insert culture dish. The co-cuhure system were divided into 3 groups：THP-1 ＋ U266 group, THP-1 ＋ U266^APEL siRNA group and the control group （THP-1 cells）. Osteoclast-like cells were identified through tartrate-resistant acid phosphatase （TRAP） staining. The mRNA levels of Cathepsin K and V-ATPase were ex- amined by RT-PCR. Osteoclast bone resorption was measured by pit assay. Results U266 cells transfected with Ad5v2 APE1 siRNA had significantly lower protein expression of APE1 and RANKL than the untransfected ceils. THP-1 cells differentiated into the osteoclast-like cells with the elevated mRNA expression of Cathepsin K and V-ATPase （ P 〈 0. 05 ）. The number of osteoclast-like cells was decreased significantly in the THP-1 ＋ U266APE1 siRNA group, as well as the mRNA levels of Cathepsin K and V-ATPase （P 〈 0. 05 ）. Gonclusion In- hibiting APE1 expression in U266 cells in a co-culture system suppresses the differentiation of THP-1 cells into osteoclast-like cells, which may be related to APE1 down-regulating RANKL in U266 cells.

关 键 词：APE1 骨髓瘤骨病 共培养 THP-1 RNA干扰 

分 类 号：R394-33[医药卫生—医学遗传学] R730.23[医药卫生—基础医学]