国际肿瘤学杂志

国际肿瘤学杂志 ›› 2017, Vol. 44 ›› Issue (12): 881-885.doi: 10.3760/cma.j.issn.1673-422X.2017.12.001

• 论著 •    下一篇

硫普罗宁对人白血病裸鼠模型IL-2治疗免疫增敏的实验研究

肖华平,谢辉,罗春阳,李庆,方玉江   

  1. 423000 郴州，湘南学院附属医院肿瘤防治中心（肖华平、谢辉、罗春阳、李庆）；美国密苏里大学医学院Ellis Fischel肿瘤中心（肖华平、方玉江）
  • 出版日期:2017-12-08 发布日期:2017-12-01
  • 通讯作者: 肖华平，Email: xiaohuaping1@163.com E-mail:xiaohuaping1@163.com
  • 基金资助:
    湖南省卫生厅科研基金（B2011-152）；湖南省自然科学基金（14JJ3136）

Immune sensitization effect of tiopronin on IL-2 immunotherapy of human leukemia cells transplanted in nude mice

Xiao Huaping, Xie Hui, Luo Chunyang, Li Qing, Fang Yujiang   

  1. Cancer Center, Affiliated Hospital of Xiangnan University, Chenzhou 423000, China; Ellis Fischel Cancer Center, University of Missouri School of Medicine, Columbia 65212, USA
  • Online:2017-12-08 Published:2017-12-01
  • Contact: Xiao Huaping, Email: xiaohuaping1@163.com E-mail:xiaohuaping1@163.com
  • Supported by:
    Hunan Health Department Scientific Research Fund (B2011-152); Natural Science Foundation of Hunan Province of China (14JJ3136)

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摘要： 目的 探讨硫普罗宁（TIP）对人白血病KG-1细胞裸鼠移植瘤白细胞介素-2（IL-2）治疗的免疫增敏作用及其可能机制。方法取对数生长期的白血病KG1细胞(1×107个/ml)，皮下接种于45只5周龄裸鼠的左后肢腹股沟，当皮下肿瘤直径为8 mm左右时，完全随机化分为3组(n=15)：Control组（腹腔内注射磷酸盐缓冲液）、IL2组（皮下注射IL2）、IL2+TIP组(皮下注射IL2+腹腔注射TIP)。观察TIP联合IL2对人白血病KG1细胞裸鼠移植瘤的治疗效果；流式细胞术检测裸鼠外周血中自然杀伤（NK）细胞数量；硝酸还原酶法检测裸鼠外周血清中反应性氮代谢物（RNM）的产量；酶联免疫吸附试验（ELISA）检测裸鼠外周血清中肿瘤坏死因子-β（TNF-β）、γ干扰素（IFN-γ）的蛋白量；原位末端标记法（TUNEL）分析细胞凋亡情况。结果IL-2和IL-2+TIP两组均可抑制移植瘤的生长，抑瘤率分别为(54.32±4.32)%、(90.15±3.75)%，IL-2+TIP组抑制肿瘤的生长更加明显(t=11.893，P＜0.001)。Control组、IL-2组、IL-2+TIP组肿瘤重量分别为（0.95±0.05）g、（0.58±0.03）g和（0.27±0.07）g，3组间差异有统计学意义(F=52.716，P＜0.001)，IL2+TIP组的肿瘤重量较IL-2组明显减轻（P=0.008）。治疗后IL-2+TIP组NK细胞数量为（0.658±0.157）个/L，明显多于IL-2组的（0.452±0.124）个/L（P=0.021）。IL-2+TIP组RNM的产量为（42.92±4.68）μmol/ml，明显低于IL-2组的（163.38±5.49）μmol/ml（P=0.007）；IL-2+TIP组TNF-β、IFN-γ的蛋白量分别为(247.68±8.24)pg/ml和(185.61±7.58)pg/ml，明显高于IL-2组的(97.48±7.28)pg/ml(P=0.021)和(70.62±8.47)pg/ml（P=0.015）。IL-2+TIP组肿瘤细胞凋亡率为（47.38±4.25）%，明显高于IL-2组的（21.41±2.79）%（P＜0.001）。结论TIP可以通过清除RNM促进NK细胞的活性，增加NK细胞诱导的肿瘤细胞凋亡，来提高白血病细胞对IL-2免疫治疗的敏感性。

关键词: 硫普罗宁, 白血病, 白细胞介素2, KG1细胞, 免疫增敏

Abstract: ObjectiveTo investigate the effects and its possible mechanisms of tiopronin (TIP) on interleukin-2 (IL-2) immunotherapy of human leukemia KG-1 cells transplanted in nude mice. MethodsKG-1 cells (1×107/ml) in logarithmic growth phase were injected subcutaneously into the groin of the left hind leg of the 45 5-week-old nude mice. When the subcutaneous tumor diameter was about 8 mm, nude mice were randomly divided into three groups (n=15): Control group (intraperitoneal injection of phosphate buffer), IL-2 group (hypodermic injection of IL-2), IL-2+TIP group (hypodermic injection of IL-2 and intraperitoneal injection of TIP). The therapeutic effect of TIP combined with IL-2 on human leukemia KG-1 cells transplanted in nude mice was observed. The number of nature killer (NK) cells in peripheral blood of nude mice was detected by flow cytometry. Nitrate reductase assay was used to detect reactive nitric metabolite (RNM) levels in peripheral blood of nude mice. Enzyme linked immunosorbent assay (ELISA) was used to detect the levels of tumor necrosis factor-β (TNF-β) and interferon-γ (IFN-γ) in peripheral blood of nude mice. Terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) assay was used to analyze apoptosis. ResultsBoth IL2 and IL2+TIP could inhibit the growth of transplanted tumor. Compared with IL-2 group ［(54.32±4.32)%］, the tumor inhibition rate of IL-2+TIP group was (90.15±3.75)%, and its inhibition of tumor growth was more obvious (t=11.893, P＜0.001). The tumor weights of Control group, IL-2 group and IL-2+TIP group were (0.95±0.05)g, (0.58±0.03)g and (0.27±0.07)g, and there was statistically significant difference among the three groups (F=52.716, P<0.001). Compared with IL-2 group, the tumor weight of IL-2+TIP group was significantly reduced (P=0.008). The number of NK cells in IL-2+TIP group was (0.658±0.157)/L, which was significantly higher than (0.452±0.124)/L of IL-2 group (P=0.021). The concentration of RNM in IL-2+TIP group was (42.92±4.68)μmol/ml, which was significantly lower than (163.38±5.49)μmol/ml in IL-2 group (P=0.007).The concentrations of TNF-β and IFNγ in IL-2+TIP group were (247.68±8.24)pg/ml and (185.61±7.58)pg/ml, which were significantly higher than (97.48±7.28)pg/ml (P=0.021) and (70.62±8.47)pg/ml (P=0.015) in IL-2 group. The apoptotic rate of tumor cells in IL-2+TIP group was (47.38±4.25)%, which was significantly higher than (21.41±2.79)% in IL-2 group (P＜0.001). ConclusionTIP can increase the sensitivity of leukemia cells to IL-2 immunotherapy by removing RNM, promoting NK cells activity and increasing NK cells-induced tumor cell apoptosis.

Key words: Tiopronin, Leukemia, Interleukin-2, KG-1 cell, Immune sensitization