摘要
目的研究TLR9激动剂——含非甲基化胞嘧啶-鸟嘌呤二核苷酸序列的寡脱氧核苷酸（CpCODN）对非小细胞肺癌（NSCLC）患者外周血单个核细胞（PBMC）抗肿瘤免疫的影响。方法分离36例NSCLC患者PBMC和肺癌细胞，RT—PCR检测TLR9 mRNA表达。PBMC分别经空白培养、不含非甲基化胞嘧啶-鸟嘌呤二核苷酸序列的寡脱氧核苷酸（non—CpGODN）和CpGODN共培养72h。^3H—TdR掺入法测定PBMC增殖变化。流式细胞仪检测T细胞表面CD69分子变化、T细胞亚群比例以及CD8^＋T胞内IFN-γ和IL4的表达。ELISA法测定PBMC上清液IFN—α的浓度，并评价抑制性ODN和氯喹对IFN—α产生的影响。分别以自体肿瘤细胞和K562细胞为靶细胞（T），以PBMC为效应细胞（E）流式细胞仪检测不同E／T时PBMC的细胞毒活性。结果NSCLC患者PBMC表达TLR9mRNA，其表达强度（0．76±0．09）与健康者（0．77±0．09）差异无统计学意义（t=0．00，P=1．00）。CpGODN诱导肺癌患者PBMC增殖（P〈0．01），上调CD3^＋T细胞表面CD69分子表达（P〈0．01），增加CD4^＋T／CD8^＋T比值（P〈0．01），增强CD8^＋T产生IFN-γ的能力（P〈0．01）。CpGODN促进PBMC分泌IFN-α（P〈0．01），抑制性ODN和氯喹抑制CpGODN诱导产生的IFN—α。CpGODN增强PBMC对K562和自体肿瘤细胞的细胞毒活性（均P〈0．01）。结论TLR9参与调控NSCLC患者的抗肿瘤免疫，TLR9的激活效应主要包括促进PBMC活化、增殖并诱导产生IFN—α，增加PBMC中CD4^＋T的比例并促进CD8^＋T分泌IFN-γ，增强PBMC对肿瘤细胞的细胞毒活性。
Objective To assess the modulation of TLR9 on anti-tumor immune responses in peripheral blood monouclear cells （PBMC） from patients with non-small-cell lung cancer （NSCLC）. Methods PBMCs were isolated from 36 NSCLC patients. Lung cancer cells were isolated from these patients and further enriched. PBMCs were cultured in RPMI-1640 medium （ blank control group ）, and medium with cytosine guanine ollgodeoxynucleotide （CpG ODN, an TLR9 agonist） or control ODN for 72 h; and then flow cytometry was used to examine the expression of CD69 antigen on the surface of CD3 ^＋ cells, [ ^3H ]-thymidine incorporation method was used to examine the cell proliferation, and the IFN-ot level in the supematant was measured. Another PBMCs were cultured in medium with interleukin （IL）-1 and then CpG ODN, control ODN, and. CpG ODN ＋ chloroquine or inhibitory ODN were added respectively for 24 - 48 h. Then the IFN-ot in the supematant was measured. Subsets were assessed by flow cytometry and the expression of TLR9-mRNA in freshly isolated PBMC was detected by RT-PCR. The production of interferon （IFN）-α in the PBMCs was measured by ELISA. The proliferation of the PBMCs was determined by [ ^3H]- thymidine incorporation. The PBMCs co-cultured with CpG ODN and autologous lung tumor cells treated with mitomycine C were used as effector cells, and K562 cells and autologous tumor cells were used as target cells flow cytometry was used to detect the capacity of PBMCs to kill autologous lung tumor cells and K562 cells. Meanwhile we investigated the intracellular expression of IFN-γ and IL-4 in CD8 ^＋ T. Results The expression level of TLR9 of the PBMCs from patients was not significantly different from that of the PBMCs from the healthy donors. The proportion of CD69 antigen expr4essing CD3^＋ T cells of the CpG ODN group was （39.5 ± 8.9 ） %, significantly higher than those of the blank control group [ （ 8.8 ± 1.2 ） %, t = 40. 30,P = 0.00] ands control ODN group [ （ 10. 6 ± 1.0） % ,t =41.85 ,P = 0.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2008年第17期1168-1172,共5页
National Medical Journal of China
基金
上海市卫生局科技发展基金资助项目（044098）
