树突状细胞疫苗特异肿瘤多肽联合树突状细胞体外刺激淋巴细胞功能评估

doi:10.19723/j.issn.1671-167X.2021.06.015

摘要/Abstract

摘要：

目的:利用树突状细胞(dendritic cell,DC)作为抗原递呈载体,检测树突状细胞疫苗特异肿瘤多肽在体外对淋巴细胞是否有刺激增殖、促进细胞因子分泌及促进对肿瘤细胞的杀伤功能。方法:外周血树突状细胞和细胞因子诱导的杀伤细胞(cytokine-induced killer,CIK)分离及培养采用贴壁法;采用CCK-8法检测淋巴细胞增殖功能;酶联免疫斑点法检测淋巴细胞的细胞因子分泌功能;CCK-8法检测淋巴细胞对肿瘤细胞的杀伤功能。实验分为肿瘤多肽组(肽+DC-CIK)、DC-CIK组和单纯CIK组进行各项功能检测与比较。结果:在白介素-2(interleukin-2,IL-2)存在时, 3组细胞均增殖明显。肿瘤多肽组在第4天、第6天(第4天Z=-3.79, P<0.001;第6天Z=-2.95, P<0.01)时,450 nm光密度显著高于CIK组,在第4天时,450 nm光密度显著高于DC-CIK组(Z=-2.02, P<0.05)。培养体系中无IL-2时,各组淋巴细胞均增殖缓慢,各时间点光密度差异无统计学意义。肿瘤多肽刺激组多种细胞因子产生量高于单纯CIK组,其中白介素-4(interleukin-4,IL-4)(Z=-2.61, P<0.01)、巨噬细胞集落刺激因子(granulocyte-macrophage colony-stimulation factor, GM-CSF)(Z=-3.85, P<0.001)、干扰素-γ(interferon-γ, IFN-γ)(Z=-3.56, P<0.001)和肿瘤坏死因子-α(tumor necrosis factor-α, TNF-ɑ, Z=-3.40, P<0.001)的分泌量与单纯CIK组相比,差异均有统计学意义。肿瘤多肽刺激组与DC-CIK组相比,除IL-4分泌量差异有统计学意义外(Z=-2.15, P<0.05), 其余各项细胞因子分泌量差异均无统计学意义。DC-CIK组与单纯CIK组相比,IFN-γ(Z=-2.44, P<0.05)、TNF-ɑ(Z=-2.26, P<0.05)和GM-CSF(Z=-3.73, P<0.001)分泌量差异有统计学意义。肿瘤多肽组与DC-CIK组、单纯CIK组在18 h与24 h的杀伤效率虽然差异无统计学意义,但有高于其他两组的趋势。结论:树突状细胞疫苗特异肿瘤多肽联合树突状细胞体外可提高CIK细胞的增殖活性,提高多个细胞因子的分泌量。

关键词: 树突状细胞, 肿瘤多肽, CIK细胞, 增殖, 细胞因子, 杀伤

Abstract:

Objective: To assess the activation function of specific tumor polypeptide for dendritic cell vaccine on lymphocytes proliferation, production of cytokines and killing activity in vitro by using dendritic cells as antigen presenting vector. Methods: Peripheral blood dendritic cells (DC) and cytokine-induced killer (CIK) were isolated and cultured by adherent culture method; CCK-8 method was used to assess the proliferation function of lymphocytes and the killing function of lymphocytes to tumor cells; enzyme-linked immunospot assay method was used to evaluate the secretion function of cytokines. The experiment was divided into tumor polypeptide group (peptide with DC-CIK), DC-CIK group and CIK group. Results: With presence of interleukin-2 (IL-2) in the culture system, the lymphocyte proliferation of the three groups was obvious. The absorbance at 450 nm of tumor polypeptide group was significantly higher than that of CIK group at the time points day 4 and day 6 (day 4: Z=-3.79, P<0.001; day 6: Z =-2.95, P< 0.01). The absorbance at 450 nm of group tumor polypeptide was significantly higher than that of DC-CIK group on day 4 (Z=-2.02, P<0.05). Without IL-2 in the culture system, lymphocytes proliferated slowly in all the three groups, and there was no significant difference in 450 nm absorbance at each time point. The levels of IL-4 (Z=-2.61, P< 0.01), granulocyte-macrophage colony-stimulation factor (GM-CSF, Z=-3.85, P< 0.001), interferon- γ (IFN- γ, Z=-3.56, P< 0.001) and tumor necrosis factor-α (TNF-ɑ, Z=-3.40, P< 0.001) of tumor polypeptide group were higher than those of CIK group. There was no significant difference in the production of cytokines except IL-4 (Z=-2.15, P< 0.05) when tumor polypeptide group was compared with DC-CIK group. The production of IFN-γ (Z=-2.44, P<0.05), TNF-ɑ (Z=-2.26, P< 0.05) and GM-CSF (Z=-3.73, P< 0.001) in DC-CIK group were higher than those of CIK group. Although there was no significant difference in killing activity between tumor polypeptide group, DC-CIK group and CIK group at hour 18 and hour 24, and the killing activity of tumor polypeptide group was higher than that of the other two groups. Conclusion: Tumor peptide combined with dendritic cells can improve the proliferation activity of CIK cells in vitro, and increase the secretion of several cytokines.

Key words: Dendritic cells, Tumor polypeptides, CIK cells, Proliferation, Cytokines, Cytotoxicity

中图分类号:

R73-36+2

杨朵,周心娜,王硕,王小利,袁艳华,杨化兵,耿会珍,彭兵,李子博,李彬,任军. 树突状细胞疫苗特异肿瘤多肽联合树突状细胞体外刺激淋巴细胞功能评估[J]. 北京大学学报（医学版）, 2021, 53(6): 1094-1098.

YANG Duo,ZHOU Xin-na,WANG Shuo,WANG Xiao-li,YUAN Yan-hua,YANG Hua-bin,GENG Hui-zhen,PENG Bing,LI Zi-bo,LI Bin,REN Jun. Assessment of lymphocytic function in vitro stimulated by specific tumor polypeptide combined with dendritic cells[J]. Journal of Peking University (Health Sciences), 2021, 53(6): 1094-1098.

图/表 4

表1

图1

图2

图3

参考文献 15

[1]	黄芸, 高建鹏, 王辉. 肿瘤疫苗临床研究评价及进展[J]. 肿瘤学杂志, 2019, 25(3):196-201.
[2]	沈季敏, 刘欣. 基于树突状细胞的肿瘤免疫治疗临床应用方式的研究进展[J]. 临床血液学杂志, 2018, 31(1):62-65.
[3]	邵小钧, 席庆. 精准医疗: 肿瘤诊治的新模式[J]. 现代肿瘤医学, 2016, 24(4):675-677.
[4]	Jang GY, Kim YS, Lee SE, et al. Improvement of DC-based vaccines using adjuvant TLR4-binding 60S acidic ribosomal protein P2 and immune checkpoint inhibitors[J]. Cancer Immunol Immunother, 2021, 70(4):1075-1088. doi: 10.1007/s00262-020-02759-6
[5]	Jiang N, Qiao G, Wang X, et al. Dendritic cell/cytokine-induced killer cell immunotherapy combined with s-1 in patients with advanced pancreatic cancer: A prospective study[J]. Clin Cancer Res, 2017, 23(17):5066-5073. doi: 10.1158/1078-0432.CCR-17-0492 pmid: 28611200
[6]	Calmeiro J, Carrascal MA, Tavares AR, et al. Pharmacological combination of nivolumab with dendritic cell vaccines in cancer immunotherapy: An overview[J/OL]. Pharmacol Res, 2021, 164: 105309[2021-01-01]. https://doi.org/10.1016/j.phrs.2020.105309.
[7]	Perez CR, De Palma M. Engineering dendritic cell vaccines to improve cancer immunotherapy[J/OL]. Nat Commun, 2019, 10(1): 5408[2021-01-01]. https://doi.org/10.1038/s41467-019-13368-y.
[8]	黄聪, 白丽. 树突状细胞肿瘤疫苗及其联合应用的研究进展[J]. 细胞与分子免疫学杂志, 2019, 35(12):1141-1145.
[9]	Zhou JY, Alvarez CA, Cobb BA, Integration of IL-2 and IL-4 signals coordinates divergent regulatory T cell responses and drives therapeutic efficacy[J/OL]. Elife, 2021, 10: e57417(2021-02-22)[2021-03-01]. https://doi.org/10.7554/eLife.57417.
[10]	于甜, 胡小鹏, 祝小红, 等. 干扰素及肿瘤坏死因子抗病毒作用研究进展[J]. 中国免疫学杂志, 2014, 30(3):409-415.
[11]	马守宝, 林丹丹, 刘海燕. 炎症细胞因子在肿瘤微环境中的作用及其作为治疗靶点的研究进展[J]. 生命科学, 2016, 28(2):182-191.
[12]	林雨虹, 吕合作, 吴俊英. GM-CSF/IL-3/IL-5的免疫调节作用[J]. 中国免疫学杂志, 2019, 35(4):500-504.
[13]	姜南雁, 唐隽, 徐云升. GM-CSF在肿瘤免疫治疗中的作用最新研究进展[J]. 免疫学杂志, 2015, 31(8):717-722.
[14]	吴晋楠, 陈建荣, 陈金亮, 等. 肺癌肿瘤微环境中白细胞介素的研究进展[J]. 临床肺科杂志, 2019, 24(12):2280-2283.
[15]	李琬琼, 高艳锋. 抗肿瘤多肽药物研究进展[J]. 药学进展, 2019, 43(10):759-766.

相关文章 15

[1]	蒋青,张雨. 新形势下运动损伤特点及细胞生物治疗的应用前景和挑战[J]. 北京大学学报（医学版）, 2021, 53(5): 828-831.
[2]	马向波,张学武,贾汝琳,高颖,刘洪江,刘玉芳,李英妮. 外周血淋巴细胞亚群检测在系统性硬化症治疗中的应用[J]. 北京大学学报（医学版）, 2021, 53(4): 721-727.
[3]	王子乔,刘燕鹰,张霞,刘田,任立敏,沈丹华,王屹,栗占国. 17例误诊为IgG4相关疾病患者的临床特点及误诊原因分析[J]. 北京大学学报（医学版）, 2019, 51(6): 1025-1031.
[4]	谢静,赵玉鸣,饶南荃,汪晓彤,方滕姣子,李晓霞,翟越,李静芝,葛立宏,王媛媛. 3种口腔颌面部来源的间充质干细胞成血管内皮分化潜能的比较研究[J]. 北京大学学报（医学版）, 2019, 51(5): 900-906.
[5]	朱红林,杜倩,谌威霖,左晓霞,李全贞,刘思佳. 系统性硬化症血清细胞因子表达谱变化及调控机制[J]. 北京大学学报（医学版）, 2019, 51(4): 716-722.
[6]	张帆,燕太强,郭卫. Rasfonin抑制骨肉瘤细胞143B的增殖和迁移[J]. 北京大学学报（医学版）, 2019, 51(2): 234-238.
[7]	刘佳兴,胡贵平,赵琳,张永明,王丽,贾光,刘瑞祥,冯慧敏,徐华东. 铬酸盐低水平长期职业接触与劳动者早期健康效应[J]. 北京大学学报（医学版）, 2019, 51(2): 307-314.
[8]	季兰岚,郝燕捷,张卓莉. 原发性骨髓纤维化引起的继发性痛风1例[J]. 北京大学学报（医学版）, 2018, 50(6): 1117-1119.
[9]	王子成，程立，吕同德，苏黎，林健，周利群. 炎症因子预处理的脂肪干细胞可明显抑制外周血单个核细胞增殖[J]. 北京大学学报(医学版), 2018, 50(4): 590-594.
[10]	唐旭，赵卫红，宋琴琴，殷华奇，杜依青，盛正祚，王强，张晓威，李清，刘士军，徐涛. SOX10对前列腺癌细胞增殖及侵袭的影响[J]. 北京大学学报(医学版), 2018, 50(4): 602-606.
[11]	汪晓彤，饶南荃，方腾姣子，赵玉鸣，葛立宏. 乳牙牙髓干细胞CD146阳性/阴性细胞亚群生物学特性的比较[J]. 北京大学学报(医学版), 2018, 50(2): 284-292.
[12]	刘颖君，欧阳翔英，王宇光，吕培军，安娜. 生长停滞特异性蛋白6在牙龈卟啉单胞菌脂多糖诱导内皮细胞黏附因子及趋化因子表达中的作用[J]. 北京大学学报(医学版), 2018, 50(1): 20-25.
[13]	陈玮, 胡凡磊, 刘洪江, 徐丽玲, 李英妮, 栗占国. 类风湿关节炎患者髓系来源的抑制细胞促进自身B细胞增殖[J]. 北京大学学报(医学版), 2017, 49(5): 819-823.
[14]	蔡燚，郭浩，李汉忠，王文达，张玉石. 结节性硬化症细胞株TSC2-/- MEFs和正常细胞株TSC2+/+ MEFs微小RNA表达谱的差异分析[J]. 北京大学学报(医学版), 2017, 49(4): 580-584.
[15]	杨迪，许珺辉，邓芙蓉，郭新彪. 纳米银对皮肤细胞半通道活性的影响及其在细胞增殖抑制中的作用[J]. 北京大学学报(医学版), 2017, 49(3): 371-375.

Metrics

Viewed

Full text

Abstract

Cited

Shared

Discussed

Serial number	Gender	Age/years	Clinical diagnosis and stage	Number of tumor specific polypeptides^*	Polypeptide target organ^#	Target cell^△
1	Female	58	NSCLC stage Ⅳ	8	Lung	A549
2	Male	59	Gastric cancer stage Ⅳ	4	Stomach	SGC-7901 BGC-823
3	Female	57	Gastric cancer stage Ⅳ	6	Stomach	SGC-7901 BGC-823
4	Male	64	Bladder cancer stage Ⅳ	3	Bladder	TCCSUP
5	Female	73	Mammary cancer stage Ⅳ	5	Breast	MCF-7
6	Female	56	NSCLC stage Ⅳ	6	Lung	A549
7	Male	60	NSCLC stage Ⅳ	6	Lung	A549
8	Female	46	NSCLC stage Ⅳ	8	Lung	A549
9	Female	73	Pancreatic cancer stage Ⅳ	5	Pancreas	CFPAC-1
10	Female	53	TNBC stage Ⅳ	6	Breast	MDA-MB-453
11	Male	46	Colonic metastasis of gastric cancer stage Ⅳ	6	Colon	HCT-116