【作者】 李娜；

【导师】 周红；

【作者基本信息】 江苏大学 ， 临床检验诊断学， 2009， 硕士

【摘要】 目的:探讨单核细胞株THP-1细胞表面膜联蛋白A2（ANXA2）在抗磷脂综合征（APS）患者IgG/β₂糖蛋白I（β₂GPI）复合物诱导细胞组织因子（TF）表达中的作用。方法:①构建插入人ANXA2 cDNA序列的真核表达质粒pIRES₂-eGFP-ANXA2,酶切及测序鉴定,脂质体介导法转染293T细胞,获得ANXA2过表达细胞。②软件分析并设计合成四条ANXA2特异性shRNA序列,与慢病毒载体pGCSIL-GFP连接,PCR及测序鉴定正确后,与过表达质粒共转染293T细胞,筛选出有效干扰序列并包装293T细胞获得重组慢病毒LV-RNAi-ANXA2,感染单核细胞株THP-1,验证内源干扰效率,建立ANXA2 RNAi细胞模型。③利用纯化的APS患者IgG与β₂GPI复合物刺激单核细胞株THP-1、ANXA2表达沉默的THP-1细胞及过表达ANXA2的293T细胞一定时间,观察不同细胞TF mRNA水平及TF活性变化,分析ANXA2在此过程中的作用。④将THP-1细胞膜裂解物与β₂GPI、抗β₂GPI抗体共沉淀,Western blotting检测共沉淀标本中ANXA2的存在,观察天然状态下的ANXA2能否与β₂GPI结合。结果:①构建的质粒pIRES₂-eGFP-ANXA2酶切及测序均正确,转染293T细胞后,ANXA2 mRNA和蛋白表达明显增高。②成功构建人ANXA2 siRNA慢病毒表达质粒并筛选出有效干扰片段;将其包装293T细胞后收获的干扰慢病毒滴度为3×10⁹ TU/ml;干扰病毒感染THP-1细胞后ANXA2的mRNA和蛋白表达均被沉默,其感染THP-1细胞的最佳MOI值为100。③APS IgG与β₂GPI复合物能够增强THP-1细胞TF mRNA及活性的表达,而对ANXA2表达沉默后的THP-1细胞TF表达没有促进作用;另外,此复合物对转染了pIRES₂-eGFP-ANXA2的293T细胞TF表达具有增强效应,证明ANXA2在此过程中起重要作用。④THP-1细胞膜裂解物与β₂GPI、抗β₂GPI抗体的共沉淀标本,经Western blotting检测证明有ANXA2的存在,说明细胞表面ANXA2能够与β₂GPI/抗β₂GPI复合物结合。结论:ANXA2作为APS IgG/β₂GPI复合物在THP-1细胞表面的受体,介导后者刺激细胞表达TF,在抗磷脂综合征（APS）高凝状态的形成中发挥重要作用。更多还原

【Abstract】 Objective:To explore the functions of annexin A2（ANXA2） in antiphospholipid syndrome patients’ IgG（APS IgG）/β₂-glycoproteinⅠ（β₂GPI）-induced tissue factor（TF） expression on monocyte drived THP-1 cells.Methods:①The pIRES₂-eGFP-ANXA2 eukaryotic expression vector carrying human ANXA2 cDNA was constructed and analyzed by restriction and sequencing.The recombinant pIRES₂-eGFP-ANXA2 plasmid was transfected into 293T cells in the mediation of liposome to get the high-ANXA2-expression cells.②Four different short hairpin RNAs（shRNA） targeting ANXA2 gene were cloned into pGCSIL-GFP vector and identificated with PCR and sequencing.The effective interference sequence was selected by Western blotting.The recombinant vector was co-transfected into 293T cells to get RNAi lentivirus LV-RNAi-ANXA2.Then the lentivirus harvested from 293T cells were transferred into THP-1 cells to block ANXA2 expression on cells.③The purifiedβ₂GPI and IgG from antiphospholipid syndrome（APS） patients were incubated in media with THP-1 cells,LV-RNAi-ANXA2-transferred THP-lcells,and 293T cells transfected with pIRES₂-eGFP-ANXA2 for a certain time.TF mRNA and TF activity on these cells were investigated.④THP-1 lysates was incubated withβ₂GPI and immunoprecipitated using anti-β₂GPI antibody,and the coprecipitates was identified by Western blotting with anti-ANXA2 antibody,in order to analyze the interaction ofβ₂GPI with ANXA2.Results:①Eukaryotic expression vector pIRES₂-eGFP-ANXA2 was correctly constructed and analyzed by restriction and sequencing. ANXA2 expression was highly increased both at mRNA and protein levels on 293T cells after transfection.②The RNA interference（RNAi） sequences targeting human ANXA2 were successfully inserted into the lentiviral vector and the high-performance RNAi sequence was sieved out. The recombinant lentivirus was harvested from 293T cells with a viral titer of 3×10⁹ TU/ml.THP-1 cells transfected with LV-RNAi-ANXA2 showed almost lockout of ANXA2 both at mRNA and protein levels.The best MOI for THP-1 cells was 100.③APS IgG/β₂GPI compound significantly increased TF mRNA expression and TF activity on THP-1 cell.While down-regulation of ANXA2 in THP-1 cells decreased TF mRNA and activity levels with the similar stimulation.293T cells transfected with pIRES₂-eGFP-ANXA2 and stimulated byβ₂GPI/anti-β₂GPI compound,could express high level of TF mRNA.④ANXA2 was detected in the immune complexes of THP-1 cells lysates by co-immunoprecipitation with anti-β₂GPI andβ₂GPI,indicating thatβ₂GPI can interact directly with ANXA2. Conclusions:ANXA2 acts as the receptor of APS IgGβ₂GPI compound,medicates TF expression on THP-1 cells induced by this compound,which is contributed to the thrombotic diathesis in APS.更多还原