人肝癌相关抗原SMP30重组慢病毒对人外周血树突状细胞成熟的影响
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摘要
目的探讨人肝癌相关抗原衰老标记蛋白30(SMP30)对人外周血树突状细胞(DC)成熟的影响。方法用Ficoll密度梯度离心法从健康成人外周血中分离获得外周血单个核细胞,用粒细胞-巨噬细胞集落刺激因子和白细胞介素4诱导生成DC,用流式细胞术对DC进行鉴定。将DC随机分为SMP30慢病毒组、空载慢病毒组,分别用人肝癌相关抗原SMP30重组慢病毒和空载慢病毒进行体外转染,另设空白对照组。荧光显微镜下观察各组绿色荧光蛋白表达情况,以判断DC的转染效率; Western blotting法检测各组SMP30蛋白表达; ELISA法检测各组白细胞介素12(IL-12)、干扰素γ(INF-γ)分泌情况;流式细胞术检测各组表面共刺激分子CD80和CD86表达。结果荧光显微镜下观察到SMP30慢病毒组和空载慢病毒组成功表达绿色荧光蛋白,而空白对照组DC无绿色荧光蛋白表达。与空载慢病毒组、空白对照组比较,SMP30慢病毒组SMP30表达高,IL-12、INF-γ分泌量高,CD80、CD86表达率高(P均<0. 05)。结论体外转染人肝癌相关抗原SMP30慢病毒可促进人外周血DC的成熟。
Objective To investigate the effects of human hepatoma-associated antigen senescence marker protein 30( SMP30) on the maturation of human peripheral blood dendritic cells( DCs). Methods Peripheral blood mononuclear cells were isolated from healthy adult peripheral blood by Ficoll density gradient centrifugation. DCs were induced by granulocyte-macrophage colony-stimulating factor and interleukin-4. We identified the DCs by flow cytometry. DCs were randomly divided into the SMP30 lentivirus group and empty vector group,which were transfected with human hepatoma-associated antigen SMP30 recombinant lentivirus and empty lentivirus,respectively; and a blank control group was also set up. The expression of green fluorescent protein in each group was observed under fluorescence microscope to determine the transfection of DCs. The expression of SMP30 in DCs of each group was detected by Western blotting. The secretion of IL-12 and INF-γ in DCs of each group was detected by ELISA. Flow cytometry was used to detect the expression of co-stimulatory molecules CD80 and CD86 on DCs surface. Results Green fluorescent protein was successfully expressed in DCs of the SMP30 lentivirus group and empty lentivirus group under fluorescence microscope. However,the expression of green fluorescent protein was not observed in DCs of the blank control group. Compared with the empty lentivirus group and the blank control group,the SMP30 lentivirus group had higher expression of SMP30,higher secretion of IL-12 and INF-γ,and higher expression rates of CD80 and CD86( all P < 0. 05). Conclusion In vitro transfection of human hepatoma-associated antigen SMP30 lentivirus can promote the maturation of human peripheral blood DCs.
Objective To investigate the effects of human hepatoma-associated antigen senescence marker protein 30( SMP30) on the maturation of human peripheral blood dendritic cells( DCs). Methods Peripheral blood mononuclear cells were isolated from healthy adult peripheral blood by Ficoll density gradient centrifugation. DCs were induced by granulocyte-macrophage colony-stimulating factor and interleukin-4. We identified the DCs by flow cytometry. DCs were randomly divided into the SMP30 lentivirus group and empty vector group,which were transfected with human hepatoma-associated antigen SMP30 recombinant lentivirus and empty lentivirus,respectively; and a blank control group was also set up. The expression of green fluorescent protein in each group was observed under fluorescence microscope to determine the transfection of DCs. The expression of SMP30 in DCs of each group was detected by Western blotting. The secretion of IL-12 and INF-γ in DCs of each group was detected by ELISA. Flow cytometry was used to detect the expression of co-stimulatory molecules CD80 and CD86 on DCs surface. Results Green fluorescent protein was successfully expressed in DCs of the SMP30 lentivirus group and empty lentivirus group under fluorescence microscope. However,the expression of green fluorescent protein was not observed in DCs of the blank control group. Compared with the empty lentivirus group and the blank control group,the SMP30 lentivirus group had higher expression of SMP30,higher secretion of IL-12 and INF-γ,and higher expression rates of CD80 and CD86( all P < 0. 05). Conclusion In vitro transfection of human hepatoma-associated antigen SMP30 lentivirus can promote the maturation of human peripheral blood DCs.
引文
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[14]López-Rela o J,Martín-Adrados B,Real-Arévalo I,et al. Monocyte-derived dendritic cells differentiated in the presence of lenalidomide display a semi-mature phenotype,enhanced phagocytic capacity,and Th1 polarization capability[J]. Front Immunol,2018,9:1328.
[2]Fujita T,Shirasawa T,Maruyama N. Expression and structure of senescence marker protein-30(SMP30)and its biological significance[J]. Mech Ageing Dev,1999,107(3):271-280.
[3]Yamaguchi M. Role of regucalcin in maintaining cell homeostasis and function(review)[J]. Int J Mol Med,2005,15(3):371-389.
[4]罗国容,谢小薰,陈维平,等.衰老标记蛋白-30——一种新的肝细胞癌相关抗原[J].广西医科大学学报,1999,16(2):3-5.
[5]赵飞兰,晁耐霞,李日伦,等.肝癌相关抗原Kinectin蛋白致敏树突状细胞诱导的CTL对肝癌细胞株杀伤作用的检测[J].中国现代医学杂志,2011,21(26):3203-3207,3212.
[6]郭晋宏,黄荣师,张瑶尧,等.肝癌相关抗原SMP30慢病毒重组子的构建及其对DC成熟作用的观察[J].世界最新医学信息文摘,2018,18(38):1-5.
[7]Jo o C,Célia G,Amílcar F,et al. Dendritic cell-based immunotherapy:a basic review and recent advances[J]. Immunologic research,2017,65(4):798-810.
[8]Wargowski E,Johnson LE,Eickhoff JC,et al. Prime-boost vaccination targeting prostatic acid phosphatase(PAP)in patients with metastatic castration-resistant prostate cancer(m CRPC)using Sipuleucel-T and a DNA vaccine[J]. J Immunother Cancer,2018,6(1):21.
[9]Ni M,Hoffmann JM,Schmitt M,et al. Progress of dendritic cellbased cancer vaccines for patients with hematological malignancies[J]. Expert Opin Biol Ther,2016,16(9):1113-1123.
[10]Aravindaram K,Wang PH,Yin SY,et al. Tumor-associated antigen/IL-21-transduced dendritic cell vaccines enhance immunity and inhibit immunosuppressive cells in metastatic melanoma[J].Gene Ther,2014,21(5):457-467.
[11]Cui J,Cui L,Liu Q,et al. Dendritic cells transfected with lentiviral vector-encoding h TERT peptide augment antitumor T cell response in vitro[J]. Mol Med Rep,2012,5(1):103-107.
[12]Kim JT,Liu Y,Kulkarni RP,et al. Dendritic cell-targeted lentiviral vector immunization uses pseudotransduction and DNA-mediated STING and c GAS activation[J]. Sci Immunol,2017,2(13):1-27.
[13]Rossetti M,Cavarelli M,Greqori S,et al. HIV-derived vectors for gene therapy targeting dendritic cells[J]. Adv Expl Med Biol,2012,762:239-261.
[14]López-Rela o J,Martín-Adrados B,Real-Arévalo I,et al. Monocyte-derived dendritic cells differentiated in the presence of lenalidomide display a semi-mature phenotype,enhanced phagocytic capacity,and Th1 polarization capability[J]. Front Immunol,2018,9:1328.