CpG ODN对非小细胞肺癌患者化疗后免疫功能的影响

The effect of CpG ODN on immune function of non-small cell lung cancer patients after chemotherapy

目的测定非小细胞肺癌(non-small cell lung cancer,NSCLC)患者化疗前后外周血单个核细胞(peripheral blood mononuclear cell,PBMC)经胞嘧啶-磷酸-鸟嘌呤寡脱氧核苷酸(cytosine-phosphorothioateguanine oligodeoxynucleotides,CpG ODN)刺激后,CD4+CD25highTreg细胞及IFN-γI、L-12的变化,评价不同时间点CpG ODN的干预效果。方法选取NSCLC患者20例,分别抽取患者化疗前(d0)、化疗后第3天(d3)及第7天(d7)外周血3 mL,分离PBMC。每份PBMC均分为实验组及对照组,实验组给予CpG ODN 2006刺激,对照组不做干预。经培养48 h后收集细胞以流式细胞仪检测CD4+CD25highTreg细胞百分比,并取上清液检测INF-γ和IL-12(pg/mL)。结果实验组CD4+CD25highTreg细胞百分比在d0、d3、d7分别为(2.22±0.66)%,(2.09±0.44)%和(2.08±0.48)%,较对照组均有明显下降(P<0.05)。实验组IFN-γ在d0、d3、d7分别为(40.64±5.90)、(40.57±5.80)和(45.30±4.78)pg/mL,d0和d7时较对照组显著增加(P<0.05),d3时变化不明显(P>0.05)。实验组IL-12在d0、d3、d7分别为(41.02±6.65)、(39.41±7.41)和(45.75±12.46)pg/mL,d0和d3时较对照组明显增加(P<0.05),而d7时变化不明显(P>0.05)。动态观察对照组,d3时CD4+CD25highTreg细胞比例下降至最低点,而后逐渐回升;实验组在d7时CD4+CD25highTreg细胞比例仍持续下降;实验组的IFN-γ值呈现升高趋势,d7和d0、d3相比明显升高(P<0.05);而实验组IL-12值在化疗前后无明显变化,即化疗前d0与化疗后d3、d7比较,以及d3与d7比较,差异均不明显(P值均>0.05)。肺癌患者化疗次数及外周血淋巴细胞的数目与CpG ODN对CD4+CD25highTreg细胞比例及IFN-γI、L-12的分泌作用无显著相关性(P>0.05)。结论 CpG ODN能有效下调CD4+CD25highTreg细胞,刺激INF-γ的分泌,优化肺癌患者抗肿瘤的免疫微环境,有利于机体的抗肿瘤免疫;肺癌患者即使化疗后外周血淋巴细胞的数目明显减少,CpG ODN仍能下调CD4+CD25highTreg细胞和刺激INF-r的分泌,发挥抗肿瘤作用。

Objective To examine the changes of CD4＋CD25high Treg cell,IFN-γ and IL-12 in cytosine-phosphorothioate guanine oligodeoxynucleotides（CpG ODN） stimulated peripheral blood mononuclear cell（PBMC） of patients with non-mall cell lung cancer（NSCLC） after chemotherapy,and to observe the dynamic changes of CD4＋CD25high Treg cell and cytokine at different time points,so as to evaluate the intervention effects of CpG ODN. Methods Twenty NSCLC patients were enrolled,and their peripheral blood samples was drawn on day 0（d0）,day 3（d3） and day 7（d7）,from which PBMC was separated and cultured for 48 hours.Every PBMC sample was divided into experiment and control group.Experiment group received CpG ODN 2006 stimulation.After 48-hour culture,both groups were sent into FACS to test the proportion of CD4＋CD25high Treg cell.ELISA was used to detect the content of INF-γ and IL-12 in the supernatants. Results The CD4＋CD25high Treg in PBMC of experiment group were（2.22±0.66）%,（2.09±0.44）% and（2.08±0.48）% on d0,d3 and d7,respectively.On d0,d3 and d7,CD4＋CD25high Treg proportion of experiment group were significantly lower than the control group（P0.05）.IFN-γ of experiment group on d0,d3 and d7 were（40.64±5.90）,（40.57±5.80） and（45.30±4.78） pg/mL,respectively.IFN-γ of experiment group were elevated significantly than the control group on d0 and d7（P0.05）,while on d3,there was no significant difference.The IL-12 of experimental group on d0,d3 and d7 were（41.02±6.65）,（39.41±7.41） and（45.75±12.46） pg/mL,respectively.The IL-12 of experiment group were elevated significantly than the control group on d0 and d3（P0.05）.In control group,the proportion of CD4＋CD25high Treg on d3 was the lowest,and ascended gradually thereafter,while in experiment group,it was still in a descending trend on d7.In experiment group,there was no significant difference in the CD4＋CD25high Treg proportion between d0 and d3,d0 and d7,or d3 and d7（P0.05）.In experiment group,IFN-γ ascended as time going on,and on d7 it was significantly higher than d0 and d3（P0.05）.In experiment group,the CD4＋CD25high Treg proportion,IFN-γ and IL-12 had no correlation with lymphocyte and chemotherapy times that NSCLC patients received. Conclusions CpG ODN could optimize the anti-tumor microenvironment of NSCLC patients,and the effect would be stronger if it is used after the chemotherapy.