APE1/Ref-1在外周血单核细胞破骨样分化中的作用研究  

Effect of APE1/Ref-1 on differentiation of peripheral blood mononuclear cells into osteoclast-like cells

作　　者：杜佳[1] 谢家印[1] 张亮[1] 周立为[1] 杨宇馨[1] 王东[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：《重庆医学》2013年第34期4112-4114,共3页Chongqing medicine

基　　金：国家自然科学基金资助项目(81172258)

摘　　要：目的探讨脱嘌呤/脱嘧啶核酸内切酶(APE1)在核因子κB受体活化因子配体(RANKL)及巨噬细胞集落刺激因子(M-CSF)诱导外周血单个核细胞(PBMCs)破骨样分化过程中的作用。方法采用密度梯度法分离提取人PBMCs;将构建的APE1siRNA表达载体导入分离获得的单个核细胞中。抗酒石酸酸性磷酸酶(TRAP)染色检测是否为破骨样细胞(OCL),蛋白免疫印记法(Western blot)检测单个核细胞APE1蛋白表达,逆转录聚合酶链式反应(RT-PCR)检测OCL中组织蛋白酶K(cathepsin K,CK)及V-ATPase基因表达水平。结果 APE1siRNA可明显降低PBMCsAPE1蛋白表达,与未感染APE1siRNA细胞相比,差异有统计学意义(P<0.05);在RANKL/M-CSF作用下单个核细胞可分化为TRAP阳性的OCL,CK及V-ATPase的表达在基因水平升高;但经APE1siRNA处理后,诱导分化的OCL数量减少,CK和V-ATPase的表达在基因水平降低。结论 PBMCs经RANKL/M-CSF诱导培养后可产生大量OCL,APE1siRNA能明显抑制单个核细胞的破骨样分化,APE1参与调节单个核细胞破骨样分化过程。Objective To investigate the effect of APE1 on differentiation of peripheral blood mononuclear cells into osteoclast-like cells（OCL） which induced by macrophage colony stimulating factor （RANKL） and macrophage colony stimulating factor （M- CSF）. Methods Human peripheral blood mononuclear cells（PBMCs） were collected by density gradient separation^Constructed APE1 siRNA expression vector Ad5v-APE1 siRNA was used to transfect PBMCs. Tartrate-resistant acid phosphatase （TRAP） method was conducted to identify the cells,the expression level of APE1 was detected by Western blot,the mRNA expression levels of Cathepsin K（CK） and V-ATPase were detected by RT-PCR. Results PBMCs transfected with APE1 siRNA had significantly lower protein expression of APE1 than untransfected cells （P^0.05） ~ PBMCs could differentiate into OCL under the stimulation of RANKL and M-CSF,the mRNA expression levels of CK and V-ATPase increased ; After APE1 siRNA treatment,the number of OCL was reduced and the levels of CK and V-ATPase mRNA decreased. Conclusion PBMCs can differentiate into a large number of OCL induced by RANKL and M-CSF, APEI siRNA significantly inhibited differentiation of PBMCs into osteoclast-like cells, APE1 may be involved in the regulation of osteoclast-like differentiation process.

关 键 词：单核细胞 细胞分化 破骨细胞 脱嘌呤 脱嘧啶核酸内切酶 

分 类 号：R733.3[医药卫生—肿瘤]