CVC1302通过小鼠骨髓源树突状细胞对免疫反应的调控

Regulation of immune response by CVC1302 through mouse bone marrow-derived dendritic cells

为获得C57BL/6小鼠骨髓源树突状细胞(DC)的体外制备方法并探讨免疫增强剂CVC1302对DC免疫调控的影响,取8周龄的C57BL/6小鼠骨髓细胞,在体外经过重组鼠源粒细胞/巨噬细胞集落刺激因子(Recombinant mouse granulocyte-macrophage colony-stimulating factor, rm GM-CSF)诱导分化为DC。诱导当天与诱导第3 d、第7 d时,用倒置显微镜观察细胞形态;诱导第7 d时,收获细胞,鉴定表型。利用流式细胞术评价CVC1302对DC表面分子表达水平的影响,用超高分辨率显微镜评价CVC1302对DC递呈抗原的影响,采用流式细胞术检测T淋巴细胞的活化数量,利用酶联免疫吸附试验(Enzyme linked immunosorbent assay, ELISA)方法检测T淋巴细胞活化后干扰素γ(IFN-γ)的分泌水平。结果表明,体外诱导的DC在显微镜下具有非常典型的树突状细胞形态,流式细胞术检测结果表明,经典的1型树突状细胞(Conventional type 1 dendritic cell, cDC1)和经典的2型树突状细胞(Conventional type 2 dendritic cell, cDC2)亚群均可检测到。CVC1302能够显著促进DC表面分子活化并且增强DC对鸡卵清白蛋白(OVA)抗原的递呈;CVC1302能够显著活化T淋巴细胞并且提高T淋巴细胞活化后IFN-γ的分泌水平。本研究利用rm GM-CSF成功在体外刺激诱导DC的产生,并证实了CVC1302在体外同样具有促进DC成熟、DC对OVA抗原的递呈及T淋巴细胞活化的能力。

To establish an effective method for acquiring bone marrow-derived dendritic cells(DC)from C57BL/6 mice in vitro and explore the immune modulation of immunopotentiator CVC1302 on DC,the bone marrow cells were isolated from C57BL/6 mice of eight week-age and were induced by recombinant mouse granulocyte-macrophage colony-stimulating factor(rm GM-CSF)in vitro to differentiate into DC.The cell morphology was observed by inverted microscope on the induction day and day 3,day 7 of induction,and cells were collected and the phenotype was identified on day 7 of induction.The expression level of DC surface molecules and antigen presentation of DC affected by CVC1302 were evaluated by flow cytometry and ultra-high resolution microscopy respectively.The numbers of activated T lymphocyte cells and secretion level of interferon-γ(IFN-γ)after activation of T lymphocyte cells were detected by flow cytometry and enzyme linked immunosorbent assay(ELISA)respectively.The results showed that the DC induced in vitro had very typical dendritic cell morphology.Moreover,subpopulations of conventional type 1 dendritic cell(cDC1)and conventional type 2 dendritic cell(cDC2)could both be detected by flow cytometry.CVC1302 could significantly promote the activation of DC surface molecules and enhance the presentation ability of DC to ovalbumin(OVA)antigen,and CVC1302 could activate T lymphocyte cells significantly and increase the secretion level of IFN-γafter activation of T lymphocyte cells.The rm GM-CSF could successfully stimulate DC production in vitro,and CVC1302 also had the ability to promote DC maturation,OVA antigen presentation by DC and activation of T lymphocyte cells in vitro.