反应性氮代谢物及其清除剂影响NK细胞抗K562细胞效应的实验研究

发布时间：2018-06-29 08:20

  本文选题：反应性氮代谢物 + 反应性氧代谢物　； 参考：《福建医科大学》2008年硕士论文

【摘要】： [目的]:探讨反应性氮代谢物(RNM)对NK细胞抗K562细胞活性的影响及硫普罗宁和还原型谷胱苷肽逆转ROM、RNM抑制NK细胞抗K562细胞活性的免疫佐剂作用。 [方法]:1.在NK细胞、K562细胞培养体系中,加入外源性ONOO-,观察RNM和ROM产量、NK细胞活性变化;2.采用IL-2及PHA为激活剂,使RNM、ROM产量增多,观察KIR和NK细胞活性的相应变化,然后在MO+NK+K562(E/T=10/1,E/MO=10/2)细胞培养体系中加入不同浓度的硫普罗宁、还原型谷胱苷肽和二氢氯组胺,定期检测RNM、ROM产量、KIR及IL-6、TNF-β、IFN-γ的产量。 [结果]:1.在NK细胞、K562细胞培养体系中,加入外源性ONOO-后,K562细胞数量和NK细胞活性显著降低,而RNM、ROM产量增加。2.①在MO细胞培养体系中,随着MO细胞数量的增加,RNM、ROM产量有所上升;而经IL-2/PHA激活后,在MO=10组, RNM从81.8898±0.7874umom/l升至91.0308±0.8956umom/l , ROM从193.1685±9.6505U/ml升至356.4275±5.9642U/ml (P0.05)。②在NK+K562混合培养体系中,加入IL-2/PHA后,当E/T =10/1时,RNM略有增加,而ROM从58.6326±2.5141U/ml增加到141.915±8.2593U/ml(P0.05),KIR从79.98%升至93.29%(P0.05);当按E/MO=10/2、10/5、10/10三种比例加入MO细胞后,RNM、ROM产量随着MO细胞数量的增加而增加,而IL-6、TNF-β、IFN-γ的产量和KIR则相反。对K562细胞数量作偏相关分析,RNM与KIR的负相关系数大于ROM(分别为r= -0.8511,-0.7141)。③在IL-2/PHA+NK+MO+K562混合培养体系中,加入TIP、GSH、DHT后,RNM产量从73.7390±3.7908 umom/l分别降至64.5516±2.1570umom/l、63.0222±2.3090 umom/l、73.1052±1.4528 umom/l,ROM产量从321.5488±10.5030 U/ml分别降至55.1665±7.0950 U/ml、54.6136±5.9515 U/ml、108.6608±6.2110U/ml,而KIR则从65.78%分别上升至84.47%、84.77%、79.56%。随着各药物浓度的增加,RNM、ROM产量逐渐减少,而IL-6、TNF-β、IFN-γ产量和KIR则逐渐升高,KIR与RNM、ROM产量呈明显负相关(P0.01)。在清除RNM、ROM,提高NK细胞抗K562细胞活性方面,硫普罗宁与还原型谷胱苷肽作用相似(P0.05),但均优于二氢氯组胺(P0.05)。 [结论]:1.外源性ONOO-对NK、K562细胞有毒性,并可转化及诱导细胞产生ROM。2.MO细胞是ROM、RNM的最主要来源,而K562细胞、NK细胞也产生少量的ROM、RNM.。3.RNM、ROM均可使NK细胞的抗瘤(抗K562)活性下降。4.硫普罗宁、还原型谷胱苷肽均可通过清除ROM、RNM保护NK细胞,提高NK细胞抗瘤活性,且对ROM、RNM的清除表现出一定的量效关系,但二氢氯组胺不能清除RNM。5.在清除ROM、RNM及提高NK细胞对K562细胞的抑制率方面,硫普罗宁、还原型谷胱苷肽效应相当,但均优于二氢氯组胺,且毒副作用较低,有望成为新的免疫佐剂用于白血病的过继性免疫治疗中。
[Abstract]:Objective: to investigate the effects of reactive nitrogen metabolites (RNM) on the anti-K562 cell activity of NK cells and the effect of tipronine and reduced glutathione on reversing the inhibitory effect of RNM on NK cell anti-K562 cell activity. [method]: 1. In K562 cell culture system, exogenous ONOO was added to observe the change of NK cell activity in RNM and ROM production. Using IL-2 and PHA as activators, the production of RNMN ROM was increased, the corresponding changes of KIR and NK cell activity were observed, and then different concentrations of tiopronin, reduced glutathione and dihydrochlorohistamine were added to MO NK K562 cell culture system. The yield of RNMN ROM and the yield of IL 6 TNF- 尾 and IFN- 纬 were detected regularly. [result]: 1. In K562 cell culture system, the number of K562 cells and the activity of NK cells decreased significantly after the addition of exogenous ONOO-, while RNMN ROM production increased .2.1 in MO cell culture system, and the ROM production increased with the increase of MO cell number. After activation of IL-2 / PHA, in MO10 group, RNM increased from 81.8898 卤0.7874umom/l to 91.0308 卤0.8956umom/l, ROM from 193.1685 卤9.6505U / ml to 356.4275 卤5.9642U / ml (P0.05). In NK K562 mixed culture system, after adding IL-2 / PHA, RNM increased slightly when E / T was 10 / 1. However, the ROM increased from 58.6326 卤2.5141U / ml to 141.915 卤8.2593U / ml (P0.05) from 79.98% to 93.29% (P0.05). When added to MO cells according to the ratio of E / MO10 / 10 / 210 / 5 / 10 / 10, the ROM output increased with the increase of MO cell number, whereas the output of IL-6TNF- 尾 -IFN- 纬 increased with the increase of MO cell number, whereas the yield of IL-6TNF- 尾 -IFN- 纬 increased with the increase of MO cell number. The negative correlation coefficient between RNM and KIR in K562 cells was higher than that in ROM (r = -0.8511-0.7141) in IL-2 / PHA NK MO K562 co-culture system. 鍔犲叆TIP,GSH,DHT鍚，

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