转谷氨酰胺酶3与树突细胞特异性捕获非整合素的细胞间黏附分子体外结合能力研究

In vitro binding ability of transglutaminase 3 to dendritic cell- specific intercellular adhesion molecule 3-grabbing nonintegrin

的 检测树突细胞特异性捕获非整合素的细胞间黏附分子（DC-SIGN）转染的HEK293T细胞和单核细胞来源的树突细胞（MDDC）膜表面DC-SIGN受体对转谷氨酰胺酶3（TG3）的识别和摄取能力。方法 采用脂质体转染的方法将融合有DC-SIGN基因片段及真核表达载体pGCMV-EGFP（增强绿色荧光蛋白）的质粒转染至HEK293T细胞，制备DC-SIGN-EGFP-HEK293T细胞；通过磁珠阴性分选出外周血中CD14＋单核细胞，并通过粒细胞-巨噬细胞集落刺激因子和白细胞介素4诱导获得MDDC。利用激光共聚焦显微镜及流式细胞仪检测转染的HEK293T细胞及MDDC上DC-SIGN受体对TG3蛋白的识别和摄取，并设立空白对照组（不加入Alexa Fluor-647染料标记的TG3）和阴性对照组（仅加入Alexa Fluor-647染料）。结果 TG3与HEK293T和MDDC细胞孵育3 h后，激光共聚焦显微镜及流式细胞仪均显示，TG3可以被HEK293T及MDDC细胞上DC-SIGN受体识别并结合摄取入细胞内，流式细胞仪检测显示TG3与MDDC的结合可被DC-SIGN 阻断抗体部分阻断。阴性对照组和空白对照组未见HEK393T或MDDC细胞对TG3的识别和结合。结论 TG3可以作为一种自身抗原被DC-SIGN受体识别、结合，并被介导摄取进入树突细胞。

Objective To evaluate the recognition and uptake of transglutaminase 3 （TG3） by dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin （DC-SIGN） receptors on the membrane surface of DC-SIGN-transfected human embryonic kidney （HEK） 293T cells and monocyte-derived dendritic cells （MDDCs）. Methods The eukaryotic vector pGCMV-enhanced green fluorescent protein （EGFP） containing DC-SIGN gene fragments was transfected into HEK293T cells to prepare DC-SIGN-EGFP-HEK293T cells by using liposome transfection method. CD14＋ monocytes were isolated from peripheral blood samples by magnetic bead-based negative selection, and then were induced by granulocyte-macrophage colony stimulating factor （GM-CSF） and interleukin-4 （IL-4） to prepare MDDCs. Laser confocal microscopy and flow cytometry were performed to evaluate the recognition and uptake of TG3 protein by DC-SIGN receptors on the surface of HEK293T cells and MDDCs. MDDCs treated without Alexa Fluor 647 dye-tagged TG3 served as blank control group, and those treated with Alexa Fluor 647 dye alone served as negative control group. Results After co-culture with TG3 for 3 hours, laser confocal microscopy and flow cytometry both showed that TG3 could be recognized by and uptaken through DC-SIGN receptors into HEK293T cells and MDDCs. Flow cytometry also revealed that the binding of TG3 to MDDCs could be partially blocked by DC-SIGN blocking antibodies. Neither the negative control group nor the blank control group showed the recognition and binding of TG3 to HEK293T cells and MDDCs. Conclusion TG3 can serve as a kind of autoantigen to be recognized and bound by DC-SIGN receptors, followed by uptake by dendritic cells.