摘要
目的:比较脐血来源的细胞因子诱导杀伤(cytokine-induced killer,CIK)细胞和肿瘤患者外周血来源CIK(peripheral blood-derived CIK,PB-CIK)细胞的增殖、激活型和抑制型表面标志物、耐药基因ABCG2以及干细胞转录因子表达的差异,探讨脐血来源CIK(umbilical cord blood-derived CIK,CB-CIK)细胞应用于肿瘤过继细胞免疫治疗的优越性。方法:采用Ficoll-plaque淋巴细胞分离液分离脐血及肿瘤患者外周血单个核细胞,加入细胞因子诱导培养CIK细胞。流式细胞术检测CIK细胞的增殖、免疫表型、激活型表面标志物CD28、CD27和抑制型表面标志物PD-1的表达,RT-PCR检测耐药基因ABCG2和干细胞转录因子c-Myc、Nanog、Oct-4、Sox2的表达。结果:CB-CIK细胞与PB-CIK细胞体外培养第4天时均开始增殖,第10天时CB-CIK细胞的增殖指数明显高于PB-CIK细胞[(251.52±16.76)%vs(158.00±43.19)%,P<0.05]。体外诱导培养13 d后CB-CIK细胞中CD3+CD56+细胞比例较PB-CIK细胞高[(21.20±4.82)%vs(10.06±3.46)%,P<0.05];激活型CD4+CD28+、CD4+CD27+和CD8+CD27+细胞比例在CB-CIK细胞中也明显升高[(32.40±16.81)%vs(18.65±9.23)%,(27.48±13.53)%vs(0.98±0.55)%,(41.76±13.98)%vs(2.58±2.10)%;P<0.05或P<0.01];而抑制型CD8+PD-1+细胞比例明显降低[(3.25±2.13)%vs(8.05±9.23)%,P<0.01]。此外,CB-CIK细胞与PB-CIK细胞均表达干细胞转录因子c-Myc、Nanog、Oct-4、Sox2,但两者之间无显著差异(P>0.05);而仅CB-CIK细胞表达高水平的耐药基因ABCG2。结论:CB-CIK细胞较PB-CIK细胞增殖速度快、激活型表面标志物表达水平高、抑制型表面标志物表达水平低、高表达耐药基因ABCG2,应用于肿瘤过继细胞免疫治疗有一定的优势。
Objective：To analyze the proliferation and differential expressions of activated and inhibitory surface markers, drug-resistance gene ABCG2 and stem cell transcription factors in umbilical cord blood-derived cytokine induced killer （CIK） cells or peripheral blood-derived CIK （PB-CIK） cells in cancer patients, and to explore the superiority of umbilical cord blood-derived CIK （CB-CIK） cells in adoptive cellular immunotherapy. Methods： Cord blood mononuclear cells and peripheral blood mononuclear cells in cancer patients were isolated using Ficoll-plaque density gradients and incubated with cytokines. The proliferation, immunophenotypes, activated surface markers （CD28, CD27） and inhibitory surface marker （PD-1） were analyzed by flow cytometry, and the drug-resistance gene ABCG2 or stem cell transcription factors （ c-Myc, Nanog, Oct-4 and Sox2 ） were determined by RT-PCR. Results： CB-CIK cells and PB-CIK cells started to proliferate on the fourth day in vitro, and the proliferation index of CB-CIK cells was significantly higher than that of PB-CIK cells on day 10 （／[251.52±16.76／]% vs ／[158.00±43.19／]%, P〈005／]. Thirteen days after incubation, the proportion of CD3＋CD56＋ cells in CB-CIK cells was higher than that in PB-CIK cells （／[21.20±4.82／]% vs ／[10.06±346／]%, P〈0.05）. When detecting the status of CIK cells, the proportions of activated CD4＋CD28＋, CD4＋ CD27＋ and CD8＋CD27＋ cells were significantly higher in CB-CIK cells than in PB-CIK cells （／[32.40±16.81／]% vs ／[18.65±923／]%; ／[27.48±13.53／]% vs ／[0.98±0.55／]%; ／[41.76±13.98／]% vs ／[2.58±2.10／]%,P〈0.05 or P〈0.01）, while the proportion of inhibitory CD8＋PD-1＋ cells was significantly lower in CB-CIK cells （／[3.25±2.13／]% vs ／[8.05±9.23／]%, P〈0.01）. The stem cell transcription factors （ c-Myc, Nanog, Oct and Sox2 ） were expressed both in CB-CIK cells and PB-CIK cells. Moreover, no significant differences were found between the two kinds of CIK cells. Furthermore, only CB-CIK cells expressed a high level of drug-resistance gene ABCG2 . Conclusion： Compared with the PB-CIK cells, the CB-CIK cells showed increasing proliferation rates, higher expressions of activated surface markers, and lower expression of inhibitory surface marker. Besides, drug-resistant gene ABCG2 is highly expressed in CB-CIK cells, showing several advantages in applying to adoptive cellular tumor immunotherapy.
出处
《中国肿瘤生物治疗杂志》
CAS
CSCD
北大核心
2013年第1期20-25,共6页
Chinese Journal of Cancer Biotherapy
基金
国家自然科学基金资助项目(No.81171985
No.81171986
No.812111102)~~
