ANX A2在抗磷脂抗体诱导THP-1细胞组织因子表达中的作用  

Role of ANX A2 in APL-induced expression of tissue factor in THP-1 cells

作　　者：李娜[1] 周红[1] 俞颖[2] 王婷[1] 黄宏亮[1] 石文霞[1] 王海波[1] 

机构地区：[1]江苏大学基础医学与医学技术学院,江苏镇江212001 [2]浙江省中医院,浙江杭州310006

出　　处：《中国病理生理杂志》2009年第12期2447-2453,共7页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.30370602;No.30670907);浙江省自然科学基金资助项目(No.Y206830);浙江省教育基金资助项目(No.20060746)

摘　　要：目的:构建针对人膜联蛋白A2(ANXA2)的RNA干扰(RNAi)慢病毒表达载体,转染THP-1细胞,探讨ANXA2在抗磷脂抗体(APL)诱导单核细胞组织因子(TF)表达中的作用。方法:设计4条ANXA2特异性RNAi的寡核苷酸序列,与慢病毒载体pGCSIL-GFP连接,PCR及测序鉴定正确后,Western蛋白印迹法筛选出有效干扰序列。包装293T细胞获得重组慢病毒LV-RNAi-ANXA2,感染单核细胞株THP-1,观察细胞ANXA2 mRNA和蛋白表达下降程度。再用APL/β2GPI复合物刺激干扰后的THP-1细胞,观察TFmRNA表达及TF活性变化。结果:成功构建RNAi慢病毒载体并筛选出有效干扰片段;包装293T细胞后病毒滴度为3×1012TU/L;感染THP-1后细胞ANXA2 mRNA和蛋白均被沉默。APL/β2GPI复合物刺激干扰后的THP-1细胞,其TF表达下降至基础水平。结论:构建的慢病毒表达载体能显著抑制THP-1细胞ANXA2的表达,进而影响APL诱导的TF表达,证明ANXA2在APL诱导的单核细胞表达TF过程中具有重要作用。AIM： To construct a lentiviral vector harboring RNAi sequence targeting human annexin A2 （ANX A2） and to investigate the functions of ANX A2 in antiphospholipid antibody （APL） -induced tissue factor （TF） expression in monocytes. METHODS： Four different short hairpin RNAs （shRNA） targeting ANX A2 gene were constructed and cloned into the pGCSIL - GFP vector. After identification with PCR and sequencing, the effective interference sequence was further determined by Western blotting analysis. The recombinant lentivirus LV - RNAi - ANX A2 harvested from 293T cells was transferred into THP - 1 cells and the ANX A2 mRNA and protein expression on THP - 1 cells were examined. The transferred -THP - 1 cells were stimulated by APL/β2GPI compound, and the TF mRNA and TF activity was assayed. RESULTS ： The RNAi sequences targeting the human ANX A2 gene were successfully inserted into the lentiviral vector and the high - performance RNA interference sequence was sieved out. The recombinant lentivirus was harvested from 293T cells with a viral titer of 3 × 10^12 TU/L. THP - 1 cells infected with LV - RNAi - ANX A2 showed almost lockout of ANX A2 both at mRNA and protein levels. The TF expression was also significantly decreased in the transferred - THP - 1 ceils induced by APL/β2GPI compound. CONCLUSION： The lentiviral vector constructed in the present study blocks the ANX A2 expression in THP- 1 ceils and further inhibits the TF expression induced by APL/β2GPI compound, which indicates that ANX A2 do play an important role in APL - induced TF expression on monocytes.

关 键 词：RNA干扰 慢病毒载体 膜联蛋白A2 抗磷脂抗体 组织因子 

分 类 号：R593.2[医药卫生—内科学]