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目的构建大鼠FcγRⅡB慢病毒诱导表达载体,观察其对巨噬细胞的作用。方法以大鼠肝脏mRNA为模板逆转录获得FcγRⅡB基因片段,并将其克隆入慢病毒表达质粒四环素(Tet)顺式应答元件(TRE),构建TRE-FcγRⅡB慢病毒表达重组质粒。将慢病毒表达重组质粒TRE-FcγRⅡB、慢病毒调节质粒Tet分别与慢病毒包装质粒共转染HEK293T细胞,分别包装表达病毒和调节病毒,测定表达病毒和可诱导病毒感染滴度。表达病毒和调节病毒共感染巨噬细胞,经梯度浓度强力霉素(DOX)诱导,免疫荧光技术检测巨噬细胞FcγRⅡB表达,实时定量PCR检测FcγRⅡB mRNA表达,Western blot法检测FcγRⅡB蛋白表达。经不同浓度DOX诱导后的腹腔巨噬细胞分别检测吞噬和趋化功能。结果重组质粒经PCR、酶切及测序鉴定正确。表达和调节病毒滴度均达到106转导单位/m L。巨噬细胞被病毒感染后经DOX诱导表达FcγRⅡB,免疫荧光染色结果显示,FcγRⅡB能够在巨噬细胞表达;实时荧光定量PCR和Western blot结果显示FcγRⅡB表达水平与DOX浓度呈正相关。巨噬细胞的吞噬和趋化功能与FcγRⅡB表达呈负相关。结论调节巨噬细胞FcγRⅡB的表达可抑制其吞噬和趋化功能。
Objective To construct a rat lentiviral vector FcγRⅡB and to observe its effect on macrophages. Methods FcγRⅡB gene fragment was reverse transcribed from rat liver mRNA and cloned into the lentiviral expression plasmid tet (Tet) cis-response element (TRE) to construct TRE-FcγRⅡB lentivirus expression recombinant plasmid. The lentiviral expression recombinant plasmid TRE-FcγRⅡB and lentivirus regulatory plasmid Tet were co-transfected with lentivirus packaging plasmid into HEK293T cells respectively, packaging and expression of the virus and regulate the virus, respectively, to measure the expression of virus and inducible virus titer. Macrophages were co-infected with virus and regulatory virus, induced by doxorubicin (DOX). The expression of FcγRⅡB in macrophages was detected by immunofluorescence assay. The expression of FcγRⅡB mRNA was detected by real-time PCR. The expression of FcγRⅡB protein was detected by Western blot. Phagocytosis and chemotaxis were detected by peritoneal macrophages induced by different concentrations of DOX. Results The recombinant plasmids were identified by PCR, restriction enzyme digestion and sequencing. Titers of both transduced and regulated viruses reached 106 transducers / mL. The results of immunofluorescence staining showed that FcγRⅡB could be expressed in macrophages. Real-time fluorescence quantitative PCR and Western blot showed that the expression of FcγRⅡB was positively correlated with DOX concentration. Phagocytosis and chemotaxis of macrophages were negatively correlated with the expression of FcγRⅡB. Conclusion The regulation of macrophage expression of FcγRⅡB can inhibit its phagocytosis and chemotaxis.