反应性氮代谢产物对NK细胞抗K562细胞活性的影响

Effects of reactive nitrogen metabolites on NK cell-mediated killing of K562 cells

目的探讨外源性、内源性反应性氮代谢产物（RNM）及其清除剂硫普罗宁（TIP）、还原型谷胱甘肽（GSH）、二氢氯组胺（DHT）对NK细胞抗K562细胞活性的影响。方法体外合成外源性ONOO^-，在NK＋K562细胞培养体系中，观察外源性ONOO^-及其清除剂对K562细胞抑制率（KIR）、肿瘤坏死因子α（TNF—β）和干扰素γ（IFN-γ）的含量及NK细胞活性影响。以白细胞介素2（IL-2）＋植物血凝素（PHA）激活单核细胞（MO）呼吸爆发产生内源性RNM，在MO＋NK＋K562细胞培养体系中，观察内源性RNM对NK细胞活性的影响，然后加入RNM清除剂观察RNM和NK细胞活性的变化。结果在NK和K562细胞培养体系中加入外源性ONOO^-后，NK活细胞率从（93．17±2．57）％下降到（71．87±1．02）％（P〈0．01），KIR从（67．47±2．64）％下降到（43．44±2．87）％（P〈0．01）；加入TIP、GSH和DHT后，NK活细胞率分别升高至（91．13±3．67）％（P〈0．05）、（88．03±1．46）％（P〈0．05）和（73．60±2．76）％（P〉0．05），KIR上升至（61．58±1．89）％（P〈0．05）、（60．68±2．07）％（P〈0．05）和（45．26±3．31）％（P〉0．05）。以IL-2＋PHA＋NK＋K562为对照，在NK＋K562＋MO混合培养体系中加入IL-2＋PHA，RNM含量从（82．10±6．60）μmom／L增至（193．65±5＋95）μmom／L（P〈0．01），KIR从（90．64±3．06）％下降至（61．29±2．22）％（P〈0．01）；加入TIP、GSH和DHT后，RNM含量分别降低至（91．32±6．81）μmom／L（P〈0．05）、（84．66±5．99）μmom／L（P〈0．05）和（188．92±5．00）μmom／L（P〉0．05），KIR上升至（84．31±4．56）％（P〈0．05）、（81．65±3．09）％（P〈0．05）和（72．20±4．10）％（P〈0．05）。结论外源性ONOO^-和MO呼吸爆发产生的RNM均可使NK细胞的抗K562细胞活性下降。TIP和GSH可通过清除RNM保护NK细胞，提高NK细胞抗K562细胞活性。

Objective To explore the effects of the exogenous and endogenous reactive nitrogen metabolites （RNM） as NK cell inhibitors on NK cell-mediated killing of K562 cells and the influence of Tiopronin （TIP）, glotamylcysteinylglycine （GSH） and histamine dihydroehloride （DHT） as RNM scavengers on reversing the suppressing effect of RNM. Methods The exogenous ONOO^- was administered in the NK ＋ K562 culture system, then the RNM scavengers were added in the NK ＋ K562 ＋ ONOO^- culture system, respectively. The concentrations of RNM, TNF-β and IFN-γ, K562 cell inhibition rate （KIR） and the percentage of living NK cells were examined. IL-2 ＋ PHA were used as monocyte （MO） activators in the culture system of MO ＋ NK ＋ K562. Then TIP, GSH and DHT were administered and the parameters of NK cell activity were analyzed. Results After exogenous ONOO^- was administered in NK ＋ K562 culture system, the percentage of living NK cells was decreased from （93. 17 ± 2.57 ） % to （71.87 ± 1.02） % （P 〈 0.01 ） and KIR was decreased from （67.47 ± 2.64） % to （43.44 ± 2.87） % （P 〈 0.01 ）. When TIP, GSH and DHT were administered into the systems, the percentage of living NK cells was increased to （91.13±3.67）% （P〈0.05）, （88.03±1.46）% （P〈0.05）, （73.60±2.76）% （P〉0.05）, respectively ; KIR was increased to （61.58 ± 1.89 ） % （ P 〈 0.05 ）, （ 60.68 ± 2.07 ） % （ P 〈 0.05 ） and （45.26± 3.31 ） % （ P 〉 0.05 ）, respectively. When IL-2/PHA were administered in the NK ＋ K562 ＋ MO culture system, RNM products was increased from （82.10 ±6.60）μmom/L to （193.65 ±5.95） μmom/L （P 〈 0.01 ） ;KIR was decreased from （90.64 ± 3.06 ） % to （ 61.29 ± 2.22 ） % （ P 〈 0.01 ）. When the TIP, GSH and DHT were administered in the systems, RNM products were decreased to （91.32±6.81 ） μmom/L （P 〈 0. 05）, （84. 66 _ 5.99 ）μmom/L （ P 〈 0.05 ） and （ 188.92 ± 5.00 ） μmom/L （ P 〉 0.05 ）, respectively; KIR was increased to （ 84. 31 ± 4. 56） % （ P 〈 0.05 ）, （ 81.65 ± 3.09） % （ P 〈 0.05 ） and （ 72.20 ± 4.10 ） % （ P 〈 0. 05 ）, respectively. Conclusion NK Cell-mediated killing of K562 ceils can be suppressed by exogenous and endogenous RNM administration. Both of TIP and GSH can protect NK cells by scavenging RNM and enhance the antineoplasmic activity of NK cells.