CpG ODN协同MAGE-3抗原肽致敏DC疫苗对膀胱癌细胞BIU-87的抑制作用
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摘要
目的:探讨含未甲基化胞嘧啶鸟嘌呤(CpG)序列的细菌寡核苷酸(CpG oligodeoxynucleotide,CpG ODN)免疫佐剂在增强黑色素瘤抗原基因-3(melanoma antigen gene-3,MAGE-3)抗原肽致敏DC抗膀胱肿瘤细胞的作用及其分子机制。方法:采用Ficoll法从健康志愿者HLA-A2型外周血中分离单个核细胞,常规方法诱导培养制备成熟DC。MTT法检测不同方式(MAGE-3、CpG ODN、MAGE-3+CpG ODN和无关抗原对照)致敏DC对T淋巴细胞增殖和CTL对靶细胞BIU-87的杀伤作用。CpG ODN+MAGE-3抗原肽致敏DC抗裸鼠BIU-87膀胱癌细胞移植瘤治疗7、11 d测定移植瘤质量变化,MTT和Western blotting法分别检测移植瘤细胞的增殖水平及其凋亡蛋白表达的变化。结果:CpG ODN+MAGE-3抗原肽致敏DC能够促进T淋巴细胞增殖(P<0.05),并显著提高活化T淋巴细胞的CTL对靶细胞BIU-87的杀伤活性(P<0.05)。体内实验表明,致敏DC治疗7、11 d的各组移植瘤质量均明显降低(均P<0.05),移植瘤的增殖能力下降也较明显(P<0.05);与其他致敏DC方式相比较,尤以CpG ODN+MAGE-3致敏DC组在治疗11 d时的移植瘤质量降低非常显著(P<0.01),且明显促进荷瘤小鼠脾脏单个核细胞的增殖能力(P<0.01);该组在治疗第3天起移植瘤组织Bcl-2表达水平明显降低、Bax水平明显升高(均P<0.05或P<0.01)。结论:CpG ODN能促进MAGE-3抗原肽致敏DC对膀胱癌BIU-87细胞的抑制作用,为膀胱癌DC疫苗的临床应用提供了实验依据。
Objective: To investigate the role of CpG ODN(CpG oligodeoxynucleotide) adjuvant in enhancing the anti-bladder cancer response induced by MAGE-3(melanoma antigen gene-3) antigen and its molecular mechanism. Methods: Mononuclear cells were isolated from HLA-A2 type peripheral blood of healthy donors by Ficoll method to prepare mature DC by conventional means. DC surface markers were detected by flow cytometry. MTT assay was used to detect the promotion effect of DCs sensitized by different means(MAGE-3, CpGODN, MAGE-3+CpG ODN, irrelevant control antigen) on the proliferation of T lymphocytes and the killing effect of CTL on BIU-87 tumor cells. The tumor mass of nude mice bearing BIU-87 bladder cell xenograft were examined on Day 7 and 11 after CpG ODN+MAGE-3 sensitized DC treatment. The expression of Bcl-2/Bax protein was detected by Western blotting while the proliferation level of xenograft cells was detected by MTT assay. Results: DCs sensitized by CpG ODN combined with MAGE-3 antigenic peptides could promote the proliferation of T lymphocytes and significantly enhance the killing effect of CTL on target BIU-87 cells(P<0.05). Compared with other sensitized DCs, in vivo experiments showed that 7 and 11 days after treatment, both the tumor volume and weight were significantly reduced(all P<0.05), and the proliferation ability of xenograft tumor was decreased(P<0.05). Compared withother sensitization means, CpG ODN+MAGE-3 especially exhibited obvious inhibitive effect on tumor growth on Day 11, and significantly promoted the proliferation of splenic monocytes of tumor bearing mice(P<0.01); moreover, Bcl-2 expression in xenograft tissues significantly decreased(P<0.01)while Bax expression significantly increased(P<0.05 or P<0.01)on Day 3 after treatment. Conclusion: CpG ODN can promote the inhibitory effect of MAGE-3 sensitized DC on bladder cancer BIU-87 cells, which will provide experimental basis for clinical application of DC vaccine in bladder cancer treatment.
Objective: To investigate the role of CpG ODN(CpG oligodeoxynucleotide) adjuvant in enhancing the anti-bladder cancer response induced by MAGE-3(melanoma antigen gene-3) antigen and its molecular mechanism. Methods: Mononuclear cells were isolated from HLA-A2 type peripheral blood of healthy donors by Ficoll method to prepare mature DC by conventional means. DC surface markers were detected by flow cytometry. MTT assay was used to detect the promotion effect of DCs sensitized by different means(MAGE-3, CpGODN, MAGE-3+CpG ODN, irrelevant control antigen) on the proliferation of T lymphocytes and the killing effect of CTL on BIU-87 tumor cells. The tumor mass of nude mice bearing BIU-87 bladder cell xenograft were examined on Day 7 and 11 after CpG ODN+MAGE-3 sensitized DC treatment. The expression of Bcl-2/Bax protein was detected by Western blotting while the proliferation level of xenograft cells was detected by MTT assay. Results: DCs sensitized by CpG ODN combined with MAGE-3 antigenic peptides could promote the proliferation of T lymphocytes and significantly enhance the killing effect of CTL on target BIU-87 cells(P<0.05). Compared with other sensitized DCs, in vivo experiments showed that 7 and 11 days after treatment, both the tumor volume and weight were significantly reduced(all P<0.05), and the proliferation ability of xenograft tumor was decreased(P<0.05). Compared withother sensitization means, CpG ODN+MAGE-3 especially exhibited obvious inhibitive effect on tumor growth on Day 11, and significantly promoted the proliferation of splenic monocytes of tumor bearing mice(P<0.01); moreover, Bcl-2 expression in xenograft tissues significantly decreased(P<0.01)while Bax expression significantly increased(P<0.05 or P<0.01)on Day 3 after treatment. Conclusion: CpG ODN can promote the inhibitory effect of MAGE-3 sensitized DC on bladder cancer BIU-87 cells, which will provide experimental basis for clinical application of DC vaccine in bladder cancer treatment.
引文
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[2]YANG J,LI Z H,ZHOU J J,et al.Preparation and antitumor effects of nanovaccines with MAGE-3peptides in transplanted gastric cancer in mice[J].Chin J Cancer,2010,29(4):359-364.
[3]ATANACKOVIC D,ALTORKI NK,STOCKERT E,et al.Vaccineinduced CD4+T cell responses to MAGE-3 protein in lung cancer patients[J].J Immunol,2004,172(5):3289-3296.DOI:10.1007/s00262-008-0490-9.
[4]REYNOLDS S R,ORAT Z R,SHAPIRO R L,et a1.Stimulation of CD8 T cell responses to MAGE-3 and melan A/MART-I by immunization to a polyvaient melanoma vaccine[J].Int J Cancer,1997,72(6):972-976.
[5]MAMHANDM,VAN BARN N,WEYNAMSP,et al.Tumor regressions observed in patients with metastatic melanoma treated with an antigenic peptide encoded by gene MAGE-3 and presented by HLA-AI[J].Int J Cancer,1999,80(2):219-230.DOI:10.1002/(SI-CI)1097-0215(19990118)80:2<219::AID-IJC10>3.0.CO;2-S.
[6]LI X Z,HAN Y,TIAN J,et al.Enhancement of dendritic cells with melanoma-associated antigen 3 for inducing cytotoxicity by cytotoxic T lymphocytes on bladder cancer BIU-87 cells[J/OL].Genetics Mol,2016,15(3):15039001[2018-04-06].https://www.ncbi.nlm.nih.gov/pmc/15039001.DOI:10.4238/gmr.15039001.
[7]NGUYEN H T,NGUYEN T T,WANG Y T,et al.Effectiveness of formalin-killed vaccines containing Cp G oligodeoxynucleotide1668 adjuvants against Vibrio harveyi in orange-spotted grouper[J].Fish Shellfish Immunol,2017,68(2):124-131.DOI:10.1016/j.fsi.2017.07.018.
[8]TIAN Y,LI M,YU C,et al.The novel complex combination of alum,Cp G ODN and HH2 as adjuvant in cancer vaccine effectively suppresses tumor growth in vivo[J].Oncotarget,2017,8(28):45951-45964.DOI:10.18632/oncotarget.17504.
[9]KRAMER K,SHIELDS N J,POPPE V,et al.Intracellular cleavable Cp G oligodeoxynucleotide-antigen conjugate enhances anti-tumor immunity[J].Mol Ther,2017,25(1):62-70.DOI:10.1016/j.ymthe.2016.10.001.
[10]CHEN J,TIAN X,MEI Z,et al.The effect of the TLR9 ligand Cp G-oligodeoxynucleotide on the protective immune response to radiation-induced lung fibrosis in mice[J].Mol Immunol,2016,80(1):33-40.DOI:10.1016/j.molimm.2016.11.001.
[11]SHANG N,FIGINI M,SHANG G J,et al.Dendritic cells based immunotherapy[J].Am J Cancer Res,2017,7(10):2091-2102.DOI:10.1038/cr.2016.157.
[12]ALBERT M L,SAUTER B,BHARDWAJ N,et al.Dendritic cells acquire antigen from apoptotic cells and induce class I-restricted CTLs[J].Nature,1998,392(6671):86-89.DOI:10.1038/32183.
[13]CHEN D,MELLMAN I.Oncology meets immunology:the cancerimmunity cycle[J].Immunity,2013,9(1):1-10.DOI:10.1007/s11538-016-0214-9.
[14]CAU X P,FAVRE N,MARTI N M,et al.Tumor-infiltrating dendritic cells are defective in their antigen-presenting function and inducible B7 expression in rats[J].J Immunother Cancer,2016,4(Suppl1):217-221.DOI:10.1186/s40425-016-0173-6.
[15]SALLUST O F,LANZAVE C,CHIA A.Efficient presentation of soluble antigen by culture human dendritic cells is maintained by granulocyte/marcrophage colony-stimulating factor plus interleukin-4 and down-regulated by tumor necrosis factor alpha[J].Exp Med,1994,179(4):1109-1118.
[16]SHIGETAK A S,KENJI S,KOICH I H,et al,Dendritic cell based adjuvant vaccination targeting Wilma tumorin patients with advanced colorectal cancer[J].Vaccines,2015,3(4):1004-1018.DOI:10.3390/vaccines3041004.
[17]HSU F J,BENIK E C,FAGNON I F,et al.Vaccination of patients with B-cell lymphoma using autologous antigen-pulsed dendritic cells[J].Med Sci(Basel),2014,2(2):82-97.DOI:10.3390/medsci2020082.
[18]SALGALLER M L,TJOAB A,LODGE P A,et al.Dendritic cellbased immunotherapy of prostate cancer[J/OL].Biomed Res Int,2014,2014:981434[2018-04-06].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4168152/.DOI:10.1155/2014/981434.
[19]宋东,杨晓玲,杨雪晶,等.DC-CIK免疫治疗晚期胰腺癌的临床疗效分析[J].中国肿瘤生物治疗杂志,2018,25(4):401-406.DOI:10.3872/j.issn.1007-385X.2018.04.014.
[20]HUANG A Y,GOLUMBEC K D,AHMADZADE H M,et al.The role of bone marrow-derived cells in presenting class I-restricted tumor antigen[J].J Immunol,2016,196(4):1964-1976.DOI:10.4049/jimmunol.1500541.
[21]任飞飞,李峰,李砺锋,等.定向编辑CTLA4基因的向导RNA体外合成体系的构建及其编辑效率[J].中国肿瘤生物治疗杂志,2017,24(12):1362-1369.DOI:10.3872/j.issn.1007-385X.2017.12.004.
[22]郭振红,曹雪涛.肿瘤免疫细胞治疗的现状及展望[J].中国肿瘤生物治疗杂志,2016,23(2):149-160.DOI:10.3872/j.issn.1007-385X.2016.02.001.
[23]VANTOMME V,DANTINNE C,AMRANI N,et al.Immunologic analysis of a phase I/II study of vaccination with MAGE-3 protein combined with the AS02B adjuvant in patients with MAGE-3-positive tumors[J].Cancer Immunol,2016,65(1):25-36.DOI:10.1007/s00262-015-1770-9.
[24]张丽洁,陈娟娟,徐翠香,等.基于HCA587的HLA-A2限制性表位肽的体内免疫原性研究[J].免疫学杂志,2016,32(1):25-28.DOI:10.13431/j.cnki.immunol.j.20160005.
[25]ZHANG L,JIANG G,YAO F,et al.Growth inhibition and apoptosis induced by osthole,a natural coumarin,in hepatocellular carcinoma[J/OL].PLo S One,2012,7(5):e37865[2018-04-06].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3360675/.DOI:10.1371/journal.pone.0037865.