【作者】 李瑾昱；

【导师】 焦顺昌；

【作者基本信息】 中国人民解放军军医进修学院 ， 肿瘤学， 2010， 博士

【摘要】 【目的】明确NK、T混合淋巴细胞(Mixture of NK and T lymphocytes cells,NKTm)对非小细胞肺癌(non-small-cell lung cancer, NSCLC)细胞的体外抑制作用,以及与顺铂(cisplatin, DDP)或与西妥昔单抗(cetuximab, C225)的联合应用效应,为临床治疗提供实验依据。探讨C225在NSCLC治疗中的作用机制；对抗体依赖性细胞介导的细胞毒作用(antibody-dependent cell-mediated cytotoxicity, ADCC)的影响因素进行研究,为C225对NSCLC的个体化综合治疗提供理论支持。【方法】第一部分以肺巨细胞癌PLA-801D为靶细胞,MTT法检测不同效靶比及不同作用时间下NKTm细胞对靶细胞的抑制率,并检测NKTm细胞以不同时序联合DDP对靶细胞的抑制率。第二部分：1、检测NSCLC细胞的分子特性：以A549、H1975、PLA-801D细胞为研究对象,免疫组化法检测表皮生长因子受体(epidermal growth factor receptor, EGFR)蛋白表达水平；荧光原位杂交法(Fluorescence in situ hybridization,FISH)检测EGFR基因扩增水平；测序法检测Kras基因、EGFR基因突变情况。2、CCK-8及流式细胞仪分别检测C225对3株肺癌细胞的体外抑制率及诱导凋亡情况,并通过基因芯片检测C225对A549细胞作用后基因表达的变化。3、采用CCK-8法检测C225介导NKTm细胞的ADCC效应对A549细胞的抑制率以及影响ADCC活性的因素：1) NKTm细胞用Anti-CD16单抗封闭,比较干预组与对照组NKTm细胞ADCC活性：2)将pEGFR-EGFP质粒用Nucleofector核转染技术导入A549细胞,比较EGFR蛋白表达量对ADCC活性的影响；3)基因测序法检测NKTm细胞FCGR3 A (Fc fragment of IgG, low affinityⅢa, receptor)基因多态性,比较NKTm细胞FCGR3A基因多态性对ADCC活性的影响。【结果】NKTm细胞对PLA-801D细胞具有明显的生长抑制作用,抑制率与效靶比呈正相关,作用4 h抑制率最大(P<0.05)。NKTm细胞与DDP联合应用具有协同效应,NKTm细胞应用4 h后再用DDP的协同抑制效果最强。A549、H1975、PLA-801D细胞的EGFR免疫组化结果均为弱阳性,EGFR基因扩增FISH检测为阴性。A549细胞Kras基因12密码子突变(GGT>AGT),H1975细胞EGFR基因第21外显子突变(L858R),PLA-801D细胞为Kras和EGFR基因野生型。生长抑制和凋亡诱导结果均表明,C225对A549细胞的作用比对PLA-801D细胞强(P<0.05)；而C225对H1975细胞的凋亡诱导作用比PLA-801D细胞弱(P<0.05)。C225对A549细胞作用后有关细胞周期、细胞迁移、肿瘤耐药及抗肿瘤免疫等方面的一系列基因表达可发生改变。C225联合NKTm细胞对A549细胞具有协同抑制效应。Anti-CD16抗体封闭后的NKTm细胞与C225联合应用的抑制率显著下降(P<0.05),且抑制率与单独NKTm细胞组的抑制率间无差异,表明协同效应主要依靠ADCC作用。转染pEGFR-EGFP质粒后的A549细胞EGFR表达呈强阳性,且多为膜阳性,NKTm细胞的ADCC作用对转染组的抑制率显著高于未转染组(P<0.05)。NKTm细胞FCGR3A-158V/V表型者比V／F和F／F表型者对A549细胞的抑制率高(P<0.05),而V／F表型与F／F表型间差异无统计学意义。【结论】NKTm细胞在体外能够显著抑制NSCLC细胞的生长,与DDP联合应用具有协同效应,NKTm细胞应用4 h后再用DDP的协同抑制效应最强。Kras基因12密码子突变(GGT>AGT)的A549细胞对C225比野生型PLA-801D细胞更敏感,而EGFR基因第21外显子突变(L858R)的H1975细胞对C225诱导的凋亡作用不敏感。C225作用后的A549细胞有关细胞周期、细胞迁移、肿瘤耐药及抗肿瘤免疫等方面的一系列基因表达发生变化,这可能与C225治疗NSCLC的作用机制相关。C225与NKTm细胞联合应用对NSCLC细胞具有协同抑制效应,且此效应主要依靠ADCC作用实现。C225介导NKTm细胞的ADCC活性可能与靶细胞EGFR蛋白表达水平和NKTm细胞FCGR3A基因多态性有关。更多还原

【Abstract】 【Objective】To investigate inhibitory effect of NKTm cells and combiniton effect of NKTm cells with DDP or cetuximab on NSCLC in vitro. To explore the mechanisms underlying cetuximab’s inhibitory effect on NSCLC and find the predictive biomarkers related to the activity of ADCC mediated by cetuximab. To provide evidence for individualized treatment of NSCLC.【Methods】Part one:PLA-801D cells were treated with NKTm cells alone for different time and at effect-to-target cell ratio (E/T) of 50:1、25:1、12:、6:1, and treated with NKTm cells plus DDP administered in different time sequence. Data on treatment efficacy were assayed by MTT. Part two:1. The molecular features of NSCLC cell lines were described using A549, H1975 and PLA-801D as target cells. Protein expression and gene amplification level of EGFR was detected separately using IHC and FISH assay. The Kras and EGFR gene mutations were detected by direct sequencing.2. Inhibition and apoptosis rate of the 3 NSCLC cell lines induced by cetuximab were analyzed by CCK-8 assay and flow cytometry respectively. Gene expression changes in A549 cells were identified by microarray.3. cetuximab mediated ADCC activity and the influencing factors were shown by calculating inhibition rate of A549 cells using CCK-8 assay:1) NKTm cells were blocked by Anti-CD 16 antibody, and ADCC activity was compared between treated groups and control groups; 2) pEGFR-EGFP plasmids were transferred into A549 cells by Nucleofector method to compare the ADCC activity of killing A549 cells with different EGFR expression level; 3) FCGR3A polymorphisms of NKTm cells were detected by direct sequencing, and ADCC activity was compared among NKTm cells with different genotypes.【Results】In our study, NKTm cells showed significant inhibitory effect on NSCLC cells in vitro. The inhibitory effect was positively correlated with E/T ratio. The highest inhibition rate appeared at the 4th h. NKTm cells increased the efficacy of DDP in inhibiting NSCLC, and the best synergistic effect was observed when DDP was administered 4 h after the use of NKTm cells.NSCLC cell lines of A549, H1975 and PLA-801D had low EGFR expression and negative EGFR FISH results. cetuximab had the highest inhibition and apoptosis rate in A549 carrying Kras mutation (GGT>AGT) and the lowest apoptosis rate in H1975 carrying EGFR mutation (L858R), P<0.05. cetuximab had influence on gene expression profiles of A549 cell line, relating to cell cycle, metastasis, resistance to chemotherapy and anti-tumor immunity.The combined use of cetuximab and NKTm cells showed a synergistic inhibitory effect on A549 cells. Application of Anti-CD 16 can block membrane receptor on NKTm cells resulting in lower synergistic inhibition rate (P<0.05), but there were no significant differences between the inhitition rate of Anti-CD 16 group and NKTm cells group. It was indicated that the synergistic effect is realized by ADCC activity. A549 cells transfected with pEGFR-EGFP plasmids expressed higher level of EGFR protein on membrane and were more sensitive to ADCC (P<0.05). The FCGR3A polymorphisms were correlated with ADCC activity of NKTm cells. The NKTm cells with FCGR3A-158V/V genotype had significantly higher ADCC than FCGR3A-158 V/F or F/F genotypes (P<0.05), but ADCC activity between V/F and F/F genotype had no statistical significance.[Conclusion] NKTm cells have inhibitory effect on NSCLC cells in vitro. The synergistic effect was observed when NKTm cells combinited with DDP, and optimal synergistic effect was achieved if DDP is added 4 h after NKTm cells administered. A549 cells with Kras mutation (GGT>AGT) are more sensitive to cetuximab than PLA-801D cells with wild type, while H1975 cells with EGFR mutation (L858R) are resistant to cetuximab mediated apoptosis. Influence on gene expression, relating to cell cycle, metastasis, resistance to chemotherapy and anti-tumor immunity may be the mechanisms underlying cetuximab’s inhibitory effect on NSCLC. The combined use of cetuximab and NKTm cells have synergistic inhibitory effect on NSCLC cells and the the synergistic effect is realized by ADCC activity. The ADCC effect may be affected by EGFR protein level of target cells and FCGR3A polymorphisms of NKTm cells.更多还原