脐血CD34^(+)造血干细胞来源的树突状细胞疫苗体外抗乳腺癌效果评价及优化  

Evaluation and optimization of anti-breast cancer effect of dendritic cell vaccine derived from cord blood CD34^(+) hematopoietic stem cells in vitro

作　　者：王淼 解辉平 严珂庆 崔洪飞 李伟 张志斐 金媛媛[3] WANG Miao;XIE Hui-ping;YAN Ke-qing;CUI Hong-fei;LI Wei;ZHANG Zhi-fei;JIN Yuan-yuan(College of Pharmacy,North China University of Science and Technology,Tangshan 063000,China;School of Basic Medicine,North China University of Science and Technology,Tangshan 063000,China;Institute of Medical Biotechnology,Chinese Academy of Medical Sciences,Beijing 100050,China)

机构地区：[1]华北理工大学药学院,唐山063000 [2]华北理工大学基础医学院,唐山063000 [3]中国医学科学院医药生物技术研究所,北京100050

出　　处：《中国新药杂志》2025年第4期377-385,共9页Chinese Journal of New Drugs

基　　金：中国医学科学院医学与健康科技创新工程资助项目(2021-I2M-1-070)。

摘　　要：目的:优化脐血CD34^(+)造血干细胞来源的树突状细胞(dendritic cell,DC)体外诱导培养方法,制备负载肿瘤细胞裂解物的DC疫苗并探索DC疫苗的抗乳腺癌活性。方法:免疫磁珠分选脐血单个核细胞后获得CD34^(+)造血干细胞,流式细胞术鉴定CD34^(+)造血干细胞纯度,经扩增培养和诱导分化培养获得DC,比较各因子在不同组合和不同时机加入对所获得DC表型的影响;通过流式细胞术检测DC对异硫氰酸荧光素-卵白蛋白(FITC conjugated ovalbumin,FITC-OVA)的抗原吞噬活性,CCK-8方法检测DC疫苗对异源脐血单个核细胞的刺激增殖能力,乳酸脱氢酶(lactate dehydrogenase,LDH)方法检测负载肿瘤细胞裂解物抗原的DC疫苗激活后的T细胞对乳腺癌细胞的体外杀伤活性。结果:扩增培养阶段,IMDM培养基+粒细胞-巨噬细胞集落刺激因子(granulocyte-macrophage colony stimulating factor,GM-CSF,100 ng·mL^(-1))+干细胞因子(stem cell factor,SCF,50 ng·mL^(-1))+FMS样酪氨酸激酶3(FMS like tyrosine kinase 3 ligand,Flt-3L,100 ng·mL^(-1))+血小板生成素(thrombopoietin,TPO,100 ng·mL^(-1))+10%胎牛血清(fetal bovine serum,FBS)+1%P/S条件培养组与IMDM培养基+GM-CSF(100 ng·mL^(-1))+SCF(50 ng·mL^(-1))+10%FBS+1%P/S条件培养组相比,收获的DC表型没有显著性差异,但前者能收获更多数量的DC;诱导分化阶段,肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)在和白介素-4(interleukin 4,IL-4)加入48 h后加入GM-CSF,比在其他时间加入收获DC的CD80和CD83的表达更高;流式细胞术结果显示未成熟DC具有较强的抗原吞噬活性;DC疫苗对异源脐血单个核细胞具有较强的刺激增殖能力;负载肿瘤细胞裂解物抗原的DC疫苗激活后的T细胞在体外对不同的乳腺癌细胞系具有显著的杀伤活性。结论:脐带血造血干细胞能够分化为数量可观的功能性DC,负载肿瘤细胞裂解物抗原的DC疫苗能激活T细胞并对乳腺癌细胞系具有显著的杀伤活性。Objective:To optimize the induction and culture method of umbilical cord blood CD34^(+) hematopoietic stem cell derived dendritic cell(DC)in vitro,prepare DC vaccine loaded with tumor cell lysate,and explore the anti-breast cancer activity of DC vaccine.Methods:CD34^(+) hematopoietic stem cells were obtained after umbilical blood mononuclear cells were sorted by immunomagnetic beads.The purity of CD34^(+) hematopoietic stem cells was determined by flow cytometry,and DC was obtained by amplification culture and induced differentiation culture.The influence of different factors added in different combinations and different times on the phenotype of the obtained DC was compared.Flow cytometry was used to detect the antiphagocytotic activity of DC on FITC-OVA,CCK-8 method was used to detect the ability of DC vaccine to stimulate the proliferation of heterologous umbilical blood mononuclear cells,and LDH method was used to detect the in vitro killing activity of T cells activated by DC vaccine loaded with tumor cell lysate antigen on breast cancer cells.Results:During amplification and culture,IMDM medium+GM-CSF(100 ng·mL^(-1))+SCF(50 ng·mL^(-1))+Flt-3L(100 ng·mL^(-1))+TPO(100 ng·mL^(-1))+10%FBS+1%P/S condition culture group and IMDM medium+GM-CSF(100 ng·mL^(-1))+SCF(50 ng·mL^(-1))+10%FBS+1%P/S,there was no significant difference in the phenotype of harvested DCs,but the former group could harvest more DCs.In the induction differentiation stage,the expression of CD80 and CD83 of the harvested DC was higher when TNF-αwas added 48 hours after the addition of GM-CSF and IL-4 than at other time.Flow cytometry showed that immature DC had strong antigen-phagocytosis activity.DC vaccine showed a strong ability to stimulate the proliferation of heterologous cord blood mononuclear cells.T cells activated by DC vaccine loaded with tumor cell lysate antigen showed significant killing activity against different breast cancer cell lines in vitro.Conclusion:Cord blood hematopoietic stem cells can differentiate into a conside

关 键 词：脐血 CD34^(+)造血干细胞 树突状细胞疫苗 乳腺癌 

分 类 号：R967[医药卫生—药理学]