【摘要】 为探讨能否通过小干扰RNA(small interference RNA,siRNA)干扰影响巨噬细胞Fcγ受体介导的吞噬作用,采用FCGRⅡA siRNA转染佛波酯(phorbol-12-myristate-13-acetate,PMA)活化人单核细胞THP-1,并设NO-TARGET、GAPDH、no-siRNA(1640)为对照。培养48～72h后,用FCM分析CD14+细胞的CD32、CD32b分布,用CD14的平均荧光强度(mean fluorescence intensity,MFI)值作为内参,得到CD32、CD32b相对MFI值;用Western blotting观察CD32a、CD32b的蛋白表达水平;进行单核细胞单层实验(monocyte monolayer assay,MMA),结合流式细胞术分析THP-1中能吞噬调理红细胞的吞噬百分比。采用配对t检验分析FCGRⅡA组与各对照组间的分布差异。Western blotting结果证实,FCGRⅡA组CD32a量明显减少(分别为NO-TARGET组、GAPDH组、1640组的22%、42%、31%),而CD32b不降低;FCM分析显示,FCGRⅡA组MFI CD32/CD14较NO-TARGET组、1640组、GAPDH组下降(P <0.05),而CD32b无明显减少;同时MMA结果显示,FCGRⅡA组吞噬细胞占比分别比NO-TARGET组、GAPDH组、1640组减少9.6%、9.2%和4.4%。提示siRNA选择性干扰可有效降低Fcγ受体CD32a表达,并降低PMA活化的THP-1吞噬能力,可作为潜在手段干扰输血反应所涉及的巨噬细胞吞噬作用。更多还原

【Abstract】 We aimed to explore the possibility of manipulating Fcγreceptor-mediated phagocytosis through siRNA interference in macrophages.siRNA of FCGRⅡA was added to treat phorbol-12-myristate-13-acetate(PMA)activated THP-1 for 48～72 h,and groups of GAPDH,NO-TARGET and no-siRNA(1640)were set in parallel as controls.FCM was used to compare MFI for CD32,CD32 bat CD14+gate with CD14 MFI as internal reference.Western blotting was used to compare CD32 a,CD32 b expression;In the meantime,monocyte monolayer assay(MMA)in combine with flow-cytometry was performed to analyze the percentages of THP-1 that had phagocytosed opsonized erythrocytes.Paired t test was utilized to check the significance between FCGRⅡAgroup and different controls.Western blotting results showed CD32 aexpression were inhibited in FCGRⅡA group(22%,42% and 31%of NO-TARGET,GAPDH and 1640 respectively)with no inhibition on CD32 bexpression;Flow data showed MFI CD32/CD14 ratio of FCGRⅡAgroup was significantly decreased than those of NO-TARGET,GAPDH and1640 groups(P < 0.05),while comparative MFI CD32 b/CD14 was almost the same as in controls.MMA results indicated the phagocytosis percentages in FCGRⅡA group was lower by 9.6%,9.2% and 4.4% respectively than those in NOTARGET,GAPDH and 1640 groups.Our results demonstrate that selectively siRNA interference of FCGRⅡAcan inhibit the CD32 aexpression of Fcγreceptor,thereby decrease the capacity of phagocytosis for THP-1 stimulated with PMA,and it may be a potential tool to manipulate the phagocytosis which is involved in transfusion reaction.更多还原