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为了探讨新型反应性氧代谢物(reactiveoxygenmetabolites,ROM)清除剂在NK细胞抑制K562细胞时作为免疫佐剂的作用,采用IL-2及PHA活化单核(MO)细胞,使MO细胞的ROM产量增加,观察NK细胞活性和K562细胞抑制率(KIR)的相应变化,然后在不同比例的MO+NK+K562细胞培养体系中加入不同浓度的ROM清除剂(硫普罗宁),定期检测ROM产量及KIR(每种检测均做3个复孔),同时应用不同浓度的二氢氯组胺(DHT)作为阳性对照。结果表明:在K562细胞、NK细胞混合培养体系中,当E/T=10/1时,加入IL-2/PHA后ROM的产量从33.17±25.02U/ml增至223.59±59.41U/ml(P<0.05),KIR从65.56%升至85.89%(P<0.05);当按E/MO=10/2、10/5、10/10三种比例加入MO细胞后,ROM产量随着MO细胞数量的增加而增加(ROM产量分别为389.79±43.83U/ml,456.74±42.77U/ml,601.42±21.92U/ml),而KIR则相反(KIR分别为82.36%,81.36%,48.09%);而在K562+NK+MO+IL-2/PHA混合培养体系中加入硫普罗宁、DHT后,当E/MO=10/2时,ROM产量从389.79±43.83U/ml分别降至-1.20±60.70U/ml和50.21±22.4U/ml(P<0.05),KIR则从82.53%分别升至96.09%和94.64%(P<0.05)。随着硫普罗宁、DHT浓度的增加,ROM产量逐渐减少。ROM产量与KIR呈负相关(r=-0.518)。当E/MO为10/5或10/10时,各浓度硫普罗宁及高浓度的DHT可使ROM产量减少(P<0.05),但KIR提高不明显(P>0.05)。硫普罗宁在提高KIR的效应方面与DHT相似(P>0.05),而在清除ROM方面,硫普罗宁较DHT为优(P<0.05)。结论:①MO细胞是ROM产生的最主要来源,其产生的ROM可使NK细胞的抗瘤(抗K562)活性下降;②新型ROM清除剂(硫普罗宁)可有效清除ROM,在一定程度上逆转ROM对NK细胞抗K562细胞的抑制作用,且在逆转ROM方面优于DHT,在提高NK细胞对K562细胞的抑制率方面效应强度与DHT相似,但毒副作用较低,有望替代DHT作为免疫佐剂用于白血病的过继性免疫治疗中。
In order to investigate the effect of novel reactive oxygen species (ROM) scavengers on NK cells as an immune adjuvant when K562 cells were inhibited, IL-2 and PHA-activated mononuclear (MO) cells were used to increase ROM production in MO cells , Observe the corresponding changes of NK cell activity and KIR inhibition rate (KIR), then add different concentrations of ROM remover (tiopronin) in different proportions of MO + NK + K562 cell culture system, regularly check ROM yield and KIR (3 replicate wells for each assay) with different concentrations of dihydrochloride histamine (DHT) as positive control. The results showed that the yield of ROM increased from 33.17 ± 25.02U / ml to 223.59 ± 59.41U / ml at the E / T = 10/1 in K562 cells and NK cells mixed culture system P <0.05). KIR increased from 65.56% to 85.89% (P <0.05). When MO cells were added at three ratios of E / MO = 10 / 2,10 / 5,10 / 10, (KIR, 82.36%, 81.36% and 48.09%, respectively) with the increase of KIR (ROM production 389.79 ± 43.83U / ml, 456.74 ± 42.77U / ml and 601.42 ± 21.92U / ml respectively) However, the yield of ROM decreased from 389.79 ± 43.83U / ml to -1.20 ± at E / MO = 10/2 after addition of tiopronin and DHT in the mixed culture system of K562 + NK + MO + IL-2 / 60.70U / ml and 50.21 ± 22.4U / ml respectively (P <0.05), KIR increased from 82.53% to 96.09% and 94.64% respectively (P <0.05). With the increase of tiopronin and DHT concentration, the yield of ROM gradually decreased. ROM production was negatively correlated with KIR (r = -0.518). When E / MO was 10/5 or 10/10, the concentration of tiopronin and high concentration of DHT could reduce the ROM yield (P <0.05), but the KIR did not increase significantly (P> 0.05). Tiopronin was similar to DHT in improving the effect of KIR (P> 0.05), while tiopronin was superior to DHT in clearing ROM (P <0.05). Conclusion: ①MO cells are the main source of ROM production, and the ROM produced by them can decrease the anti-tumor (anti-K562) activity of NK cells; ② The new ROM remover (tiopronin) can effectively remove ROM and reverse to some extent ROM on NK cell anti-K562 cells, and in the reverse ROM ROM is better than DHT, NK cells in enhancing the inhibition rate of K562 cells with similar intensity and DHT, but the toxicity is low, is expected to replace DHT as an adjuvant For leukemia in adoptive immunotherapy.