未成熟树突状细胞吞噬抗原3小时的微丝细胞骨架结合蛋白的研究
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贵州医科大学

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Study of microfilament cytoskeleton-binding proteins phagocytosis antigen in immature dendritic cells for 3 hours
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    摘要:

    目的 分析影响未成熟树突状细胞(immature dendritic cells,imDCs)行使抗原吞噬功能时的关键微丝细胞骨架结合蛋白(microfilament cytoskeleton binding proteins,MCBPs)。方法 从健康人外周血中分离单核细胞(monocytes,MOs),经细胞因子诱导培养获得imDCs和成熟树突状细胞(mature dendritic cells,mDCs);将imDCs分别与低分子量(40kD)和高分子量(150 kD)的右旋糖酐孵育1、3、6小时,正常培养的细胞作对照,流式细胞技术检测吞噬了右旋糖酐的imDCs的百分率;将imDCs与40kD的右旋糖酐孵育3小时,流式细胞技术检测imDCs的免疫表型分子,免疫荧光技术观察丝状肌动蛋白(filamentous actin,F-actin)的变化及PFN1、WASP、α-actinin在细胞中的定位,qPCR和WB分别检测MCBPs的mRNA和蛋白表达差异,利用系统生物学算法中的逐步回归和主成分分析方法筛选成分系数最大的MCBPs。结果 imDCs对低分子的抗原吞噬更快,吞噬时长维持3个小时左右;imDCs吞噬抗原3小时,细胞表型和形态逐渐向mDCs分化,并且F-actin发生了明显的重塑,其MCBPs包括PFN1、CDM、WASP、CAPZB、Filamin A、α-actinin的表达发生了下调,WAVE1、Arp2/3 复合体、Fascin的表达发生了上调;信号蛋白Rac1的mRNA表达上调,CDC42和RhoA的mRNA表达下调;免疫荧光结果显示PFN1、WASP、α-actinin在imDCs吞噬抗原过程中发生转位;PFN1的成分系数最大。结论 在imDCs行使抗原吞噬功能时PFN1可能是该过程中的关键MCBPs,这对于深入理解imDCs免疫学功能来说具有重要意义。

    Abstract:

    Objective Analysis of key filament cytoskeletal binding proteins that affect the performance of antigenic phagocytosis by immature dendritic cells Methods Monocytes (MOs) were isolated from healthy human peripheral blood and imDCs and mature dendritic cells (mDCs) were obtained by cytokine-induced culture; ImDCs were cultured with low molecular weight (40 kD) and high molecular weight (150 kD) dextrans for 1, 3 and 6 hours, respectively, and the percentage of imDCs phagocytosed by flow cytometry was detected; imDCs were incubated with 40 kD dextrose for 3 h, immunophenotypic molecules of imDCs were detected by flow cytometry; localization of microfilaments and PFN1, WASP, α-actinin in cells were observed by immunofluorescence technology; mRNA and protein expression differences of MCBPs were detected by qPCR and WB; The stepwise regression and principal component analysis methods in the systems biology algorithm were used to screen the MCBPs with the largest component coefficients. Results The antigen phagocytosis of small molecules was faster, and the engulfment time lasted about 3 hours; 3 hours after imDCs phagocytosis antigen, cell phenotype and morphology gradually differentiated to mDCs, and the microfilament cytoskeleton underwent obvious remodeling; the expression of binding proteins including PFN1, CDM, WASP, CAPZB, Filamin A and α-actinin were down-regulated, the expression of WAVE1, Arp2/3 complex and Fascin were upregulated; The mRNA expression of signaling protein Rac1 was up-regulated, and the mRNA expression of CDC42 and RhoA was down-regulated; The immunofluorescence results showed that PFN1, WASP, and α-actinin transposed during the antigen engulfment of imDCs; PFN1 had the highest component coefficient. Conclusion PFN1 may be the key MCBPs in the process of antigenic phagocytosis of imDCs, which is of great significance for understanding the immunological function of imDCs.

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  • 收稿日期:2023-06-07
  • 最后修改日期:2024-06-02
  • 录用日期:2024-06-05
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