CAJ | 학술논문

目的:构建针对人膜联蛋白A2(ANX A2)的RNA干扰(RNAi)慢病毒表达载体,转染THP-1细胞,探讨ANX A2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ANX A2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-ANX A2,感染单核细胞株THP-1,观察细胞ANX A2 mRNA和蛋白表达下降程度.再用APL/β_2GPI复合物刺激干扰后的THP-1细胞,观察TF mRNA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3×10~(12)TU/L;感染THP-1后细胞ANX A2 mRNA和蛋白均被沉默.APL/β_2GPI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ANX A2的表达,进而影响APL诱导的TF表达,证明ANX A2在APL诱导的单核细胞表达TF过程中具有重要作用.
목적:구건침대인막련단백A2(ANX A2)적RNA간우(RNAi)만병독표체재체,전염THP-1세포,탐토ANX A2재항린지항체(APL)유도단핵세포조직인자(TF)표체중적작용.방법:설계4조ANX A2특이성RNAi적과핵감산서렬,여만병독재체pGCSIL-GFP련접,PCR급측서감정정학후,Western단백인적법사선출유효간우서렬.포장293T세포획득중조만병독LV-RNAi-ANX A2,감염단핵세포주THP-1,관찰세포ANX A2 mRNA화단백표체하강정도.재용APL/β_2GPI복합물자격간우후적THP-1세포,관찰TF mRNA표체급TF활성변화.결과:성공구건RNAi만병독재체병사선출유효간우편단;포장293T세포후병독적도위3×10~(12)TU/L;감염THP-1후세포ANX A2 mRNA화단백균피침묵.APL/β_2GPI복합물자격간우후적THP-1세포,기TF표체하강지기출수평.결론:구건적만병독표체재체능현저억제THP-1세포ANX A2적표체,진이영향APL유도적TF표체,증명ANX A2재APL유도적단핵세포표체TF과정중구유중요작용.
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/β_2GPI 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 cells induced by APL/β_2GPI compound. CONCLUSION: The lentiviral vector constructed in the present study blocks the ANX A2 expression in THP-1 cells and further inhibits the TF expression induced by APL/β_2GPI compound, which indicates that ANX A2 do play an important role in APL-induced TF expression on monocytes.