融合蛋白CTP-FoxM1诱导C57BL/6小鼠骨髓来源树突状细胞的活化

Activation Analysis of C57BL/6 Mouse Bone Marrow Derived Dendritic Cells Stimulated by Fusion Protein CTP-Fox M1

树突状细胞(dendritic cells,DCs)疫苗负载的肿瘤抗原可以使DCs大量表达表面分子II类组织相容性抗原(major histocompatibility complex class-II,MHC-II)及共刺激分子CD40、CD80、CD86,促进Th1(T helper 1)型细胞因子的分泌,从而可以将肿瘤抗原充分递呈给T淋巴细胞,诱导机体产生杀伤肿瘤细胞的免疫应答。其中,肿瘤抗原是否能充分活化DCs是机体发挥抗肿瘤免疫应答的关键。该研究以融合蛋白CTP-Fox M1(cytoplasmic transduction peptide-forkhead box M1)为研究对象,探讨其对C57BL/6小鼠骨髓来源DCs的活化作用。首先,分别将胞质转导肽(cytoplasmic transduction peptide,CTP)、Fox M1(forkhead box M1)抗原相关基因连接入p COLD-TF-DNA,构建重组原核表达质粒p COLD-TF-CTP-Fox M1,转化感受态大肠杆菌BL21,IPTG(isopropylβ-Dl-thiogalactopyranoside)诱导表达,经亲和纯化、Western blot验证得到融合蛋白CTP-Fox M1。然后,用CCK-8试剂盒检测经不同浓度的融合蛋白CTP-Fox M1处理48 h后的DCs的存活率;用激光共聚焦显微镜观察融合蛋白CTP-Fox M1在DCs的定位情况;流式细胞仪检测DCs表面MHC-II类分子和共刺激分子CD40、CD80、CD86的表达情况;ELISA法检测DCs培养上清中细胞因子白介素-12(interleukin-12,IL-12)的分泌水平。结果显示:CTP-Fox M1对DCs的生长有一定的抑制作用,且与其浓度有关,浓度为1μg/m L的融合蛋白CTP-Fox M1处理组的DCs存活率(80.93%±8.36%)明显高于浓度为2μg/m L融合蛋白CTP-Fox M1处理组DCs的存活率(70.13%±5.38%,P<0.001)和4μg/m L融合蛋白CTP-Fox M1处理组DCs的存活率(62.97%±4.06%,P<0.001)。在激光共聚焦显微镜下可以观察到该融合蛋白能够在DCs胞质内定位,与对照组相比,该融合蛋白可以上调DCs表面MHCII类分子和共刺激分子CD40、CD80、CD86的表达量(P<0.01,P<0.05)及细胞因子IL-12的分泌量(P<0.001)。结果提示,融合蛋白CTP-Fox M1能促进DCs的活化,对肝癌的免疫治疗有一定的潜能,为下一步探索融合蛋白CTP-Fox M1负载的DCs是否能够在体内发挥免疫效应奠定了一定的基础。

of cell surface molecules MHC-II and costimulatory molecules such as CD40, CD80 and CD86, enhance the Thl type cytokines secretion and present the antigens to T cells to induce the anti-tumour immune response. Whether the tumour associated antigens can fully activate DCs is the key factor during the whole anti-tumour immune response. In order to detect the effect of the fusion protein CTP-FoxM1 on the activation of C57BL/6 mouse bone marrow derived DCs, CTP-FoxM1 fragments were sequentially inserted into plasmid pCold-TF, then the constructed recombinant plasmid was transformed into E.coli BL21 and induced by IPTG. The fusion protein CTP-FoxM1 was purified by affinity chromatography and finally identified by Western blot assay. The survival rates of DCs treated with different concentrations of CTP-FoxM 1 for 48 h were tested by the CCK-8 assay; the cytoplasmic localization of the fusion protein was observed by the confocal microscopy; the expressions of MHC-II, CD40, CD80 and CD86 phynotypes of DCs were evaluated by the flow cytometry and the secretion of cytokine IL-12 was detected by the ELISA assay. The result indicated that the survival rates were surpressed by the fusion protein CTP-FoxM1 in a concentration depended manner. The survival rate of 1 μg/mL of CTP-FoxM1 treated DCs （80.93%±8.36%） was higher than that of the 2 μg/mL of CTP-FoxM1 treated DCs （70.13%±5.38%, P〈0.001） and 4 μg/mL of CTP- FoxM1 treated DCs （62.97%±4.06%, P〈0.001）. The fusion protein CTP-FoxM1 was located in the cytoplasm of DCs. It could upregulate the expression levels of MHC-II, CD40, CD80 and CD86 of DCs and the secretion of IL- 12 （P〈0.001） compared with PBS treated groups. The study suggests that the fusion protein CTP-FoxM1 has the capacity of activating DCs and the potency of immunotherapy of hepatocellular carcinoma. These findings make a foundation for the further study of the immunological effects ofDCs pulesd with CTP-FoxM1 in vivo.