中文摘要：

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。

英文摘要：

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.