The characteristics and significance of non-clonal chromosome aberrations in the in vitro expanded NK cells

doi:10.3969/j.issn.2095-4344.0645

Abstract

Abstract:

BACKGROUND: The significance of non-clonal chromosome aberrations (NCCAs) in in vitro expanded stem cells has been discussed in many studies; however, it is not well documented in the expanded NK cells.
OBJECTIVE: To investigate the characteristics and significance of NCCAs in the in vitro expanded NK cells.
METHODS: Peripheral blood mononuclear cells were isolated from patients with tumors and healthy controls. Artificial antigen presenting cells were used to stimulate the proliferation of NK cells. On the 7^th day of culture, the cells were collected for phenotype identification and interferon-γ secretion test by flow cytometry and for cytogenetic analysis by the conventional G-banding technique. The difference in the frequency of NCCAs and interferon-γ secretion between the two groups and the correlation between NCCAs and interferon-γ secretion were studied.
RESULTS AND CONCLUSION: NCCAs were found in 6 of 7 samples from the tumor patient group and 6 of 12 samples from the healthy controls. In the tumor patient group, 17 metaphases containing NCCAs were found out of 481 metaphases, and in the control group, 7 NCCA-containing metaphases were detected out of 785 metaphases [3.53% (17/481) vs. 0.89% (7/785), Z=-2.212, P=0.028]. Four patients with a history of chemoradiotherapy showed higher NCCA frequencies than those patients undergoing chemoradiotherapy (P=0.034) and the healthy controls (P=0.003). No difference was found between the newly diagnosed tumor patients and the healthy controls (P=0.819). The ratios of interferon-γ secreting NK cells in the two groups were not significantly different (P=0.371) and the correlation between the frequency of NCCAs and the interferon-γ secreting level was not identified. It was the first time that the characteristics of NCCAs in the artificial antigen presenting cells-expanded NK cells and the relationship between NCCAs and the NK cell function were preliminarily studied. The effect of NCCAs on the function and survival of NK cells in vitro and in vivo needs further investigations.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Killer Cells, Natural, Chromosomes, Interferon-gamma, Tissue Engineering

CLC Number:

R394.2

Li Yu-long, Huang Zhou-feng, Zhang Lei, Wu Cheng-ye, Cheng Wei, Dong Xiao-yan, Zhu Zun-min, Sun Kai. The characteristics and significance of non-clonal chromosome aberrations in the in vitro expanded NK cells[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(33): 5362-5367.

Figures/Tables 2

2.1 NK细胞的免疫表型验证目前，CD3-CD16+CD56+被认为是NK细胞的表面标志。在培养的第7天，80.5%-91.2%的细胞呈现CD3-CD16+CD56+表型(图1)，人工化抗原递呈细胞成功刺激了NK细胞的增殖(图2)。 2.2 肿瘤组和对照组NK细胞中检出非克隆性染色体异常人数及非克隆性染色体异常发生频率比较人工化抗原递呈细胞的核型为复杂的克隆性染色体异常(图3)，而所有NK细胞样本中均未检测到克隆性染色体异常。FISH检测人工化抗原递呈细胞性别染色体为两个绿色X信号(图4)，表示K562细胞来自女性，因此异常的男性核型不可能源自人工化抗原递呈细胞污染。在7例肿瘤患者的NK细胞样本中，6例检测出非克隆性染色体异常(图5)，而在12例健康志愿者中，6例检出非克隆性染色体异常，两者相比差异无显著性意义(85.71% vs. 50%，P=0.173)。在肿瘤组中，共分析481个中期分裂相，发现含有非克隆性染色体异常的分裂相17个；在健康组中，共分析785个中期分裂相，发现含非克隆性染色体异常的分裂相7个，两者相比差异有显著性意义[3.53%(17/481) vs. 0.89% (7/785)，Z=-2.212，P=0.028]。 2.3 有/无放化疗史的个体NK细胞中非克隆性染色体异常的频率比较 4例有放化疗病史的患者，264个中期分裂相中发现含有非克隆性染色体异常的分裂相15个，明显高于对照组[5.68%(15/264)vs.0.89%(7/785)，Z=-2.991，P=0.003]及初诊肿瘤患者[5.68%(15/264) vs. 0.92%(2/217)，Z=-2.121，P=0.034]，而初诊肿瘤患者与对照组非克隆性染色体异常的频率差异无显著性意义[0.92% (2/217) vs. 0.89%(7/785)，Z=-0.229，P=0.819]，见表2。 2.4 肿瘤组与对照组NK细胞γ-干扰素表达水平比较培养后肿瘤组与对照组NK细胞表达γ-干扰素水平差异无显著性意义[(28.41±3.54)% vs. (31.88±7.50)%，F=0.846，P=0.371]，接受过放化疗的患者与对照组以及初诊肿瘤患者相比，差异亦无显著性意义[(26.4±3.21)% vs. (31.88±7.50)%，F=2.847，P=0.114；(26.4±3.21)% vs. (32.1±5.63)%，F=2.135，P=0.204]。 2.5 NK细胞中非克隆性染色体异常与γ-干扰素分泌水平相关性分析肿瘤组和对照组NK细胞中，非克隆性染色体异常与γ-干扰素表达水平均无明显相关性(r值分别为-0.472，-0.557，P值分别为0.285，0.06)。"

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