Patients with non-small cell lung cancer (NSCLC) harboring epidermal growth factor receptor (EGFR)-activating mutations can be treated with EGFR-tyrosine kinase inhibitors (TKIs). Although EGFR-TKI-targeted drugs bring survival promotion in patients with EGFR mutations, drug resistance is inevitable, so it is urgent to explore new treatments to overcome drug resistance. In addition, wild-type EGFR lacks targeted drugs, and new targeted therapies need to be explored. Ferroptosis is a key research direction for overcoming drug resistance. However, the role and mechanism of regulating ferroptosis in different EGFR-mutant NSCLC types remains unclear. In the present study, H1975 (EGFR T790M/L858R mutant), A549 (EGFR wild-type) and H3255 (EGFR L858R mutant) NSCLC cell lines were used. The expression of ferroptosis markers in these cell lines was detected using western blotting and reverse transcription-quantitative PCR. Cell viability was determined using the MTT assay and reactive oxygen species (ROS) levels were measured using flow cytometry. The results showed that, compared with EGFR wild-type/sensitive mutant cells, EGFR-resistant mutant cells were more sensitive to the ferroptosis inducer, erastin. Furthermore, the mammalian target of rapamycin (mTOR) inhibitor, everolimus (RAD001), induced cell death in all three cell lines in a dose-dependent manner. The ferroptosis inhibitor, ferrostatin-1, could reverse cell death in EGFR-resistant mutant and EGFR wild-type cells induced by RAD001, but could not reverse cell death in EGFR-sensitive mutant cells. Compared with EGFR wild-type/sensitive mutant cells, EGFR-resistant mutant cells were more sensitive to RAD001 combined with erastin. In addition, a high-dose of RAD001 reduced the expression levels of ferritin heavy-chain polypeptide 1 (FTH1), glutathione peroxidase 4 (GPX4) and ferroportin and significantly increased ROS and malondialdehyde (MDA) levels in EGFR-resistant mutant and EGFR wild-type cells. In the present study, GPX4 inhibitor only or combined with RAD001 inhibited the AKT/mTOR pathway in EGFR-resistant mutant cells. Therefore, the results of the present study suggested that inhibition of the mTOR pathway may downregulate the expression of ferroptosis-related proteins in EGFR-resistant and EGFR wild-type NSCLC cells, increase the ROS and MDA levels and ultimately induce ferroptosis.

目的探讨携带甲胎蛋白（AFP）的重组腺相关病毒（rAAV）感染树突状细胞（DCs）对肝癌细胞株SMMC-7721的体外杀伤作用,为肝癌非手术治疗提供方法。方法首先确定AFP基因序列并合成AFP基因,将其转染至重组腺相关病毒rAAV,之后将转染AFP的rAAV感染树突状细胞,并将感染rAAV的DC与SMMC-7721共同培养,同时设未感染rAAV的DC作为对照组,用MTT法检测rAAV-DC及DC对SMMC-7721的杀伤活性。结果感染rAAV的DC对SMMC-7721的杀伤活性为89.00%,DC对SMMC-7721的杀伤活性为75.19%,两者比较有差异（P＜0.05）。结论携带AFP的rAAV感染DC后,能明显增强DC对SMMC-7721的杀伤活性。

目的:探讨N端截短的羧肽酶E(N-terminal truncated carboxypeptidase E,CPE△N)在人宫颈癌中的表达情况及其对肿瘤细胞增殖迁移功能的影响.方法:采用qRT-PCR方法检测35例宫颈癌患者肿瘤组织中CPE△N的表达水平,同时检测CPEΔN高表达组肿瘤细胞增殖和迁移功能并进行相关分析.结果:宫颈癌患者肿瘤组织CPEΔN mRNA/18sRNA比值平均为2.01±0.65,显著高于对照组,其高表达情况与细胞迁移功能具有显著相关(P<0.05).结论:宫颈癌组织中CPE△N过表达并促进肿瘤细胞的迁移,由此推断CPE△N极有可能作为宫颈癌患者病情发展和预后评价的生物标志物.

目的:探讨焦磷酸溴代醇(BrHPP)诱导的γ9δ2T淋巴细胞对胃癌细胞株SGC-7901的体外杀伤作用,为胃癌的治疗提供新的模式.方法:分离人外周血单核细胞,采用BrHPP联合白细胞介素2(IL-2)、anti-human TCRγ/δ联合IL-2两种方法进行γ9δ2 T淋巴细胞的体外诱导培养,在培养的第10天用流式细胞仪分别检测两种方法培养的细胞γ9δ2 TCR等的表达,并对培养细胞上清进行IL-2、肿瘤坏死因子α(TNF-α)、干扰素γ(IFN-γ)等细胞因子检测,最后用两种方法培养的细胞对SGC-7901进行体外杀伤实验.结果:BrHPP联合IL-2培养诱导的细胞 γ9δ2TCR有较高表达为61.60％,其中 γ9TCR为65.10％,δ2TCR为64.10％;anti-human TCRγ/δ联合IL-2培养诱导的细胞γ9δ2TCR表达率较低为10.70％,其中γ9TCR为98.10％,δ2TCR为10.60％;培养细胞上清IL-2、TNF-α、IFN-γ等细胞因子表达与对照组比较有明显差异(P<0.01),但两实验组之间比较没有差异(P>0.05).BrHPP联合IL-2培养诱导的细胞对SGC-7901的杀伤活性为77.06％,anti-human TCRγ/δ联合IL-2为78.80％.结论:BrHPP联合IL-2培养诱导的细胞无论是γ9δ2TCR表达,还是IL-2、TNF-α、IFN-γ等细胞因子的分泌水平,以及对SGC-7901的体外杀伤活性都比较高,证明BrHPP联合IL-2的方法诱导的细胞具备了γ9δ2T淋巴细胞的特征和功能,为γ9δ2T淋巴细胞的进一步研究和应用奠定了基础.

目的探讨焦磷酸溴代醇（BrHPP）诱导人外周血单核细胞产生γ9δ2T淋巴细胞的方法,同时与抗γδTCR诱导产生的细胞进行比较,并用IL-2诱导的细胞作为对照,以提供一种新的诱导γ9δ2T淋巴细胞的方法,为γ9δ2T淋巴细胞的研究应用提供有效的细胞。方法分离健康人外周血单核细胞,计数后加到细胞培养瓶培养,每瓶先加入IL-2,1000IU/ml,然后按不同浓度加入BrHPP、抗γδTCR等,至培养第10天用流式细胞仪检测3种方法培养细胞γ9δ2TCR的表达。结果培养10天3种细胞γ9δ2TCR的表达分别为BrHPP:61.6%;anti-γδTCR:10.7%;IL-2:0.6%。结论 BrHHP可以诱导出高表达γ9δ2的T淋巴细胞。

Objective: To monitor the immune function of thyroid cancer patients and analyze its clinicopathological correlation. Methods: The distribution of circulating lymphocyte subpopulations and serum cytokine (IFN-γ, TNF-α, TGF-β, and IL-10) levels in 59 Papillary Thyroid Cancer (PTC) patients, 33 goiter patients, and 50 healthy controls were measured by flow cytometry and ELISA. Results: The percentages of CD3+ T lymphocytes and CD3+CD4+/CD3+CD8+ ratio in PTC patients decreased obviously. While the percentages of CD4+CD25+CD127low Tregs in PTC patients increased apparently, but it had no relationship with clinical characteristics. Serum TNF-α, IFN-γ, IL-10, but not TGF-β levels in PTC patients were significantly higher than that in healthy controls. The inclusion of these four cytokines improved the classification efficacy among the three groups. Conclusion: It is apparent that the immune function is compromised in thyroid cancer patients. Combined detection of serum cytokines could help distinguish patients with thyroid disease and healthy individuals.

目的 检测分析宫颈癌患者外周血淋巴细胞亚群的差异分布及临床意义.方法 采用流式细胞术检测宫颈癌患者和健康对照成人外周血中CD3+、CD3+CD4+、CD3+CD8+、NK、NKT、CD8+CD28+、CD8+CD28-、CD3+HLA-DR+、CD3-HLA-DR+、Treg细胞的百分比,同时结合宫颈癌患者的临床诊断分期做对比分析.结果 与健康对照组比较,宫颈癌患者外周血中CD3+(P=0.0002)和CD3+CD4+T细胞百分比(P=0.0004)以及CD3+CD4+/CD3+CD8+比值(P=0.0256)显著降低;宫颈癌患者CD3+CD8+T细胞百分比与对照组比较无显著差异,但是随着患者临床分期的提高呈降低趋势(P=0.0480).NK细胞(P＜0.0001)、活化T细胞(CD3+HLA-DR+:P＜0.0001)、活化B细胞(CD3-HLA-DR+:P=0.0021)百分比显著提高;细胞毒性T细胞(CD8+CD28+)细胞百分比降低(P=0.0006),抑制性T细胞(CD8+CD28-)百分比提高(P=0.0009);CD4+CD25brightT细胞(Treg)阳性细胞百分比与对照组比较没有显著差异,但CD4+CD25brightT细胞(Treg)在CD4+淋巴细胞中的百分比显著高于对照组(P=-0.0335).宫颈癌患者外周血中CD8+CD28-/CD8+CD28+之比与对照组比较显著提高(P＜0.0001).结论 宫颈癌患者细胞免疫功能降低.精确分析宫颈癌患者的T淋巴细胞亚群变化对于临床诊断、治疗和预后评估具有重要的实际意义.

目的 比较来自脐带血和恶性肿瘤患者外周血的细胞因子诱导的杀伤细胞(cytokine-induced killer,CIK)体外抗肿瘤作用的差异.方法 体外诱导培养肺癌患者外周血和脐血来源的CIK细胞,MTT法和流式细胞术检测诱导的CIK细胞的抗肿瘤活性及机制.结果 来自肺癌患者外周血和脐血的CIK细胞中CD3+细胞、CDYCD56+和CD3+CD8+细胞百分比均伴随培养时间的延长而升高,组间比较无统计学差异;诱导培养21 d时,脐血CIK细胞和肺癌患者外周血来源的CIK细胞的增殖率分别为(185.76±39.68)％、(254.32±74.60)％,二者之间差异有统计学意义(P=0.0012);脐血CIK细胞的抗肿瘤活性高于肺癌患者外周血来源的CIK细胞(效靶比10:1,靶细胞为K562细胞时,P=0.0016;靶细胞为A549细胞时,P=0.0022);脐血CIK细胞CD107a表达高于来自肺癌患者外周血的CIK细胞(靶细胞为K562细胞时,P=0.0469;靶细胞为A549细胞时,P=0.0413);脐血CIK细胞IFN-γ和TNF-α的分泌水平高于肺癌患者CIK细胞(P=0.0352;P=0.0389);脐血CIK细胞和肺癌患者CIK细胞诱导K562细胞凋亡的百分率为(25.30±4.87)％和(19.87±5.41)％,二者之间差异无统计学意义(P=0.2658).结论 流式细胞术可同时检测CIK细胞的表型、功能及作用机制.本研究为临床应用CIK治疗提供更多选择和实验室依据.

目的 建立标准化的实验和分析方法,采用流式细胞术比较分析癌症患者新鲜和冻存复苏后的外周血淋巴细胞亚群的差异.方法 于2014年6～12月收集肺恶性肿瘤患者静脉血样本(2 mL样本),每个样本取300μL全血用于淋巴细胞亚群分析;剩余全血经裂解红细胞后冻存(40％或90％胎牛血清).30 d后复苏细胞,流式细胞术分析淋巴细胞亚群变化.结果 复苏并培养24h后,癌症患者PBMC的存活率分别为(67.70±2.80)％(90％ FCS)和(80.47±3.32)％ (40％ FCS,P=0.0069).新鲜和冻存的的外周血CD3+、CD3+CD4+、CD3+CD8+、CD3-CD16+56+、CD3+CD16+56+和CD4+CD25+T细胞的百分率之间无显著差异(P＞0.05,n=8,40％FCS).结论 短期冻存癌症患者的PBMC可用于后续检测T淋巴细胞亚群,尤其对于晚期癌症患者而言,可避免多次采血造成的负担同时提高实验的一致性.此外,本实验建立的方法也可用于临床抗肿瘤免疫治疗和其他免疫监测.

Objective:To construct nanoparticle complexes and delivery siRNA sequence so as to inhibit SOCS1 gene expression on cord blood dendritic cells.Methods: Synthesized nanoparticle complexes using SiO2 and Fe3O4 enveloped by PEI.Using LipofectamineTM2000 as control,agarose gel electrophoresis analyzed the efficiency of nanoparticle-siRNA combination;Fluorescence microscopy and flow cytometry examined the cellular uptake of nanoparticle-siRNA complexes;Western blot assay detected the expression of SOCS1 protein before and/or after DCs were treated with nanoparticle-siRNA complexes;MTT assay evaluated the antitumor activities of effector cells stimulated by DCs.Results: Compared with the control group,nanoparticle complexes can efficiently combine the siRNA sequence.Nanoparticle-siRNA complexes were uptaked by cord blood dendritic cells.However,DCs treated with nanoparticle complexes alone insignificantly inhibit SOCS1 gene expression;in addition to the external magnetic field,SOCS1 gene expression were significantly reduced,and paralleled with the enhanced antitumor activities of effector cells stimulated by DCs.Conclusion: Nanoparticle complexes showed safety and high efficiency in siRNA sequence delivery in vitro.The expression of SOCS1 gene on cord blood dendritic cells were significantly silenced by nanoparticle-siRNA complexes.The results presented in this study provided a new way of thinking and experimental basis,which are expected to be useful for designing more effective dendritic cell-based antitumor immunotherapeutic strategies.