基于癌睾抗原CTNNA2长肽疫苗免疫活性检测

目的:设计针对癌睾抗原CTNNA2的包含多个表位的长肽,并鉴定这些长肽的免疫活性。方法:通过NETCTL1.2、SYFPEITHI和BIMAS软件预测打分选取CTNNA2的HLA-A2限制性的表位;对包含多个优势CTL表位区域选取合适长度的长肽,通过体外活性实验验证长肽是否具有免疫活性。酶联免疫斑点实验(ELISPOT)检测DC荷长肽诱导的外周血单个核细胞(PBMCs)中γ-干扰素(IFN-γ)的释放;细胞因子染色检测表位肽诱导CTL的能力;LDH法和CFSE法检测CTL对靶细胞的杀伤活性。结果:ELISPOT和细胞因子染色实验结果显示长肽P778-800、P857-880诱导的CTL具有分泌IFN-γ的能力。CFSE和LDH细胞毒实验结果显示表位P778-800、P857-880对靶细胞有一定的杀伤作用。结论:成功筛选鉴定出针对癌睾抗原CTNNA2的2条长肽,可能对基于CTNNA2的癌症免疫疗法的未来临床试验具有意义。     

不同免疫途径对抗原肽修饰DC疫苗免疫效应的影响

目的探讨不同的免疫途径对抗原肽修饰树突状细胞(DC)疫苗免疫效果的影响。方法用鸡卵清蛋白(OVA)MHCⅠ限制性多肽SIINFEKL(OVAp257-264)包被小鼠骨髓来源的DC,通过皮下、腹腔、静脉或肌肉注射免疫小鼠,7天后行体内细胞毒性T淋巴细胞杀伤实验(InvivoCTL)分析CTL杀伤活性和细胞内IFN-γ染色(ICS)分析免疫小鼠脾脏CD8+细胞产生IFN-γ的情况。结果免疫7天后,InvivoCTL结果显示SIINFEKL修饰的DC皮下、腹腔、静脉或肌肉注射免疫小鼠其特异性CTL杀伤效应分别是37.3%±7.3%、61.0%±4.2%、56.9%±3.6%和10.8%±2.3%;ICS结果示四组小鼠产生IFN-γ的CD8+细胞占总CD8+细胞的比例分别是0.31%±0.07%、0.85%±0.12%、0.76%±0.14%和0.15%±0.04%。结论不同免疫途径可明显影响抗原肽修饰DC疫苗的免疫效应,其中腹腔注射诱发的抗原特异性CTL反应最强,静脉注射者次之,而皮下注射特别是肌肉注射较弱,提示通过腹腔注射DC疫苗可能是安全、高效的免疫途径。     

食管癌中COX-2表达与其临床病理特征及预后的关系

目的：检测COX-2在食管癌中的表达，探讨其与食管癌临床病理特征及术后患者预后的关系。方法：应用SABC免疫组化染色法检测89例手术切除的食管癌组织和20例正常食管粘膜组织COX-2蛋白的表达，分析COX-2在食管癌组织的表达与患者性别、年龄、肿瘤部位、病变长度、浸润深度、区域淋巴结转移、远处转移、鳞癌分化程度的关系。并对其中81例有随访资料者，分析COX-2表达与预后的关系。结果：食管癌组COX-2呈阳性表达率为94.38%（84/89），且着色较深，正常食管组COX-2呈阳性表达率为60%（12/20），着色浅。食管癌中，随着肿瘤浸润深度的增加、鳞癌细胞分化程度的减低，COX-2的表达逐步增强（P＜0.05）。COX-2的表达与其它临床病理特征，包括性别、年龄、肿瘤生长部位、肿瘤大小、有无区域淋巴结转移、有无远处转移无相关（P＞0.05）。COX-2低表达组与高表达组的生存时间有显著差异，前者生存时间较后者为长（P＜0.01）。结论：COX-2在食管癌中的表达高于正常食管粘膜。肿瘤浸润越深、鳞癌分化程度越低，COX-2的表达越强；COX-2的表达与食管癌患者的年龄、性别、肿瘤生长部位、肿瘤长度、有无区域淋巴结转移、有无远处转移均无关。COX-2的表达与食管癌术后患者的预后相关，COX-2呈低表达者的生存时间比COX-2高表达者长。     

肺癌细胞总RNA转染的DC疫苗体外诱导肺癌患者特异性抗肿瘤免疫的实验研究

目的：为了探讨肺癌细胞总RNA转染的DC疫苗体外诱导特异性抗肿瘤免疫的能力。方法：采用分离肺癌患者外周血单核细胞体外诱导DC细胞，Trizol法提取肺癌细胞系Calu-6总RNA，用脂质体包裹总RNA转染DC并诱导特异性CTL的扩增，LDH法和ELISA法检测CTL的杀伤活性和IFN-γ分泌。结果：经肺癌细胞总RNA转染的DC特异性表面标志及功能相关分子表达均上调，转染后的DC可显著刺激异体／自体T淋巴细胞增殖，诱导的特异性CTL对携带Calu-6抗原的靶细胞的杀伤率显著高于LAK细胞，再次接触相同抗原时其IFN-γ分泌量显著增高。结论：肺癌细胞总RNA转染的DC疫苗可在体外诱导出特异性抗肿瘤免疫。     

TIL识别的HLA-A2限制性人黑色素瘤特异抗原肽的研究

目的：探讨TIL识别的人黑色素瘤HLA-A2限制性的肿瘤抗原肽特性。方法：通过亲和层析柱从三种肿瘤细胞系（624-Mel、Chap-Mel和JY）中纯化HLA-A2蛋白和结合的多肽分子，经弱酸高温处理和离心超滤后，应用反相．高效液相色谱层析（RT-RPLC）分离各多肽组份，并将其各组份与T2细胞共同孵育、进行重建TIL识别的人黑色素瘤特异表位试验，鉴定肽分子生物活性，通过质谱分析所获活性肽分子特性，参照活性肽序列，制备人工合成肽加以进一步证实。结果：从RT-RPLC层析的624-Mel的肽组份，经特异表位鉴定发现3个活性峰（P19、P25和P31）。其中P31，TIL杀伤率达67％，质谱分析表明肽分子量为948，氨基酸序列为H^＋Ala Lue Trp Lue Phe Phe Gly Val Lue OH^-的9肽分子。经特异表位测定表明人工合成肽具有纯化活性肽的生物活性特征。结论：分离鉴定的这-9肽分子是一种TIL识别的HLA-A2限制性肿瘤抗原肽，为肿瘤防治、合成肽疫苗的研究奠定了可靠的实验基础。     

抗原肽致敏白介素12基因修饰的树突状细胞免疫治疗转移性肺癌

目的　探讨肿瘤多肽致敏的白介素 12 (IL 12 )基因修饰的树突状细胞 (DC)对自发性肺转移癌的治疗作用。方法　小鼠足垫注射 3LLLewis肺癌细胞建立自发性肺转移癌模型 ,经骨髓来源的IL 12基因修饰、3LL特异抗原多肽Mut1体外致敏DC(DC IL 12 Mut1)皮下免疫 2次 ,观察荷瘤鼠肺脏质量、肺表面转移结节、存活期的变化及相应免疫指标等变化 ,实验分8组 ,组间差异行t检验 ,生存期行时序检验。结果　与对照病毒组 (DC LacZ Mut1)及未处理DC组相比 ,DC IL 12 Mut1组肺脏质量最轻 (P <0 .0 1)、肺表面转移结节最少 (P <0 .0 1)、存活期最长 (P <0 .0 1) ,其诱导CTL活性 (P <0 .0 1)和NK活性 (P <0 .0 1)最显著。结论　MHCⅠ类限制性肿瘤抗原多肽致敏的IL 12基因修饰的DC能通过诱导显著的抗肿瘤免疫反应而对自发性肺转移癌有明显的治疗作用     

癌-睾丸抗原SSX-2抗原肽的鉴定及功能分析

SSX基因又称滑膜肉瘤X断裂点基因（Synovial Sarcoma Xbreakpoint，SSX），是个多成员的基因家族，其中一些成员编码的产物被认为是癌-睾丸抗原（Cancer-Testis Antigen，CTA）。CTA是一类可在多种肿瘤组织中表达，而在睾丸以外的其它正常组织中几乎不表达的抗原。因此，CTA非常适用于肿瘤免疫治疗。目前对SSX基因家族中的SSX-2抗原的研究显示，SSX-2抗原分子作为肿瘤免疫治疗的靶物质有着潜在的应用前景。现将其研究进展综述如下。     

颗粒性肽-DNA复合疫苗抗肿瘤免疫的研究

诱导有效的抗肿瘤免疫应答是肿瘤免疫治疗的主要目标 ,由于肽疫苗和DNA疫苗具备较高的安全性和实用性 ,因而成为肿瘤疫苗学领域中发展最为迅速的肿瘤免疫治疗方案之一。但是这两种疫苗形式仍然具有各自的缺点 ,其抗原呈递动力学存在显著差异 ,提示这两种疫苗可能存在互补性。本研究对多肽疫苗和DNA疫苗进行综合 ,设计出新型肽 DNA复合疫苗 ,并以P815A为模式抗原 ,证实了该种新型疫苗激发抗肿瘤免疫的有效性     

基于幽门螺杆菌UreB优势抗原表位多抗原肽疫苗制备及其免疫保护作用

目的 构建串联式幽门螺杆菌UreB抗原优势表位UreB322和UreB527原核重组表达系统,制备UreB322和UreB527表位与多肽载体Poly-Asp-Lys的多抗原肽(MAP)疫苗并确定其免疫原性和免疫保护性.方法 采用连接引物PCR构建含肠激酶(EK)位点的串联式UreB322和UreB527表位基因及其原核表达系统.表达的目的重组融合蛋白8×[rEK-UreB322-EK-UreB527-EK]经EK水解后用Sephadex G-25柱分离rUreB322-EK和rUreB527-EK片段.采用碳二亚胺法将rUreB322-EK、rUreB527-EK与Poly-Asp-Lys载体分子交联,制备出多抗原肽疫苗MAP-rUreB322/B527.采用ELISA和Western blot分别检测各重组表位肽及MAP-rUreB322/527的抗原性和免疫反应性.采用幽门螺杆菌SS1株感染BALB/c小鼠模型检测MAP-rUreB322/527免疫保护效果.结果 获得了8次重复串联的UreB322和UreB527编码基因及其原核表达系统,目的融合蛋白8×[rEKUreB322-EK-rUreB527-EK]表达量可达细菌总蛋白的48％.肠激酶可将目的融合蛋白完全水解为rUreB322-EK和rUreB527-EK片段.rUreB322-EK和rUreB527-EK与Poly-Asp-Lys交联率高达92.5％.幽门螺杆菌全菌抗体及rUreB-IgG均能识别rUreB322-EK、rUreB527-EK和MAP-rUmB322/527并与之结合.MAP-rUreB322/527免疫小鼠血清抗体水平明显高于rUreB(P＜0.05).50或100μgMAP-rUreB322/527对幽门螺杆菌SS1株感染小鼠的保护率(83.3％和91.7％)明显高于等量rUreB(41.7％和50.0％)(P＜0.05).结论 本研究成功地构建了幽门螺杆菌UreB抗原优势表位UreB322和UreB527重复串联式编码基因及其原核表达系统,基于UreB优势抗原表位的多抗原肽疫苗MAPrUreB322/527能显著提高免疫保护效果.     

长链非编码RNA在肺癌中的研究进展

长链非编码RNA（LncRNA）是一类转录本长度超过200nt的RNA,不编码蛋白,大多数位于细胞核,以RNA的形式在多层面上（表观遗传调控、转录调控以及转录后调控等）调控基因的表达水平。其广泛参与机体的生理和病理过程,在恶性肿瘤的发生和发展中起着重要作用。     

COX-2抑制剂对肺癌裸鼠移植瘤血管生成素基因表达的影响

目的：探讨环氧化酶-2(COX-2)抑制剂尼米舒利（NIM）对肺癌裸鼠移植瘤的血管生成素基因表达的影响及意义。 方法： 人肺癌A549细胞接种于裸鼠皮下，建立肺癌裸鼠移植瘤模型并予NIM治疗，计算NIM的抑瘤率，RT-PCR检测裸鼠移植瘤组织血管生成素-1、2 (Ang-1、Ang-2) mRNA表达, 免疫组织化学法测定裸鼠移植瘤组织微血管密度(MVD)。 结果： NIM可有效抑制裸鼠移植瘤的生长，其抑瘤率为43.02%。 NIM治疗组裸鼠移植瘤组织Ang-2 mRNA水平显著低于对照组（P<0.01），Ang-1 mRNA水平无显著改变（P>0.05）, Ang-2/Ang-1 mRNA比值下降（P<0.01）；同时MVD明显低于对照组（P<0.01）。 结论： COX-2抑制剂NIM可下调Ang-2基因表达,改变Ang-2/Ang-1 mRNA比值，该作用可能是COX-2抑制剂抑制肿瘤血管生成从而抑制肿瘤生长的机制之一。     

血浆胃泌素释放肽前体，细胞角蛋白19和癌胚抗原对各型肺肿瘤的诊断价值

目的探讨胃泌素释放肽前体（ProGRP）,细胞角蛋白19（CYFRA21—1）,癌胚抗原（CEA）对各型肺癌的诊断价值。方法检测85例健康对照人群、49例肺良性疾病患者及143例各型肺癌患者血浆中的ProGRP,CYFRA21-1,CEA三个指标,并对小细胞肺癌（SCLC）患者进行ProGRP的跟踪监测。结果SCLC患者中ProGRP水平[中位数（M）179．1ng／m1）]明显高于肺腺癌（M35．3ng／m1）、鳞癌（肘33．3ng／m1）、健康对照（膨35．6ng／m1）和良性肺病（M33．3ng／m）,差异有统计学意义（P〈0．001）;ProGRP对SCLC的诊断灵敏度和特异性分别为60．6％和95．0％。SCLC治疗有效组,ProGRP降低了45．9％,SCLC疾病进展组,ProGRP升高了103．1％,差异均有统计学意义（P〈0．05）。CEA在肺腺癌转移组中水平（M10．22ng／ml）明显高于无转移组（M3．85ng／m1）和鳞癌组（M2．56ng／m1）,差异有统计学意义（P〈0．01）。结论血浆ProGRP是一个很好的SCLC诊断指标和疗效评价指标;CEA明显升高是腺癌肺外转移指标。     

NKG2D engagement of colorectal cancer-specific T cells strengthens TCR-mediated antigen stimulation and elicits TCR independent anti-tumor activity

The NKG2D receptor is expressed by human NK, gammadelta T and alpha/beta T lymphocytes and its engagement results in the stimulation of effector cells. We evaluated the role of NKG2D receptor in anti-colorectal cancer (CRC) immune response. The cell surface expression of stress-inducible NKG2D ligands MICA/B (MHC class I-related chain molecules A/B) and ULBP (UL16 binding protein) by a panel of CRC lines was evaluated by flow cytometry. MICA and ULBP2/3 were widely expressed by the analyzed lines, with a minority of them being also ULBP-1+, whereas MICB was undetectable. CD8+ and CD4+ HLA-restricted anti-tumor T cell clones of a CRC patient were used to evaluate whether NKG2D engagement could mediate tumor recognition. Three out of four CD8+ T cell clones recognized the autologous tumor with a marginal NKG2D engagement, a finding that was correlated with the weak expression of NKG2D ligands by the autologous tumor. On the contrary, NKG2D triggering of these CD8+ T cell clones induced recognition of allogeneic CRC lines showing high expression of MICA and ULBP. A costimulatory role of NKG2D was observed with one CD4+/NKG2D+ T cell clone when stimulated by tumors sharing the HLA class II alleles and expressing NKG2D ligands. Taken together these data indicate that the engagement of NKG2D, depending on the expression of its ligands by target cells, can influence the pattern of anti-tumor reactivity by T lymphocytes.     

The innate immune architecture of lung tumors and its implication in disease progression

Lung malignancies are the leading cause of cancer-related mortality. By virtue of its unique physiological function, the lung microenvironment is highly dynamic and constantly subjected to mechanical, chemical and pathogenic stimuli. Thus, the airways rely on highly organized innate defense mechanisms to rapidly protect against pathogens and maintain pulmonary homeostasis. However, in the context of lung malignancy, these defenses often provide collateral inflammatory insults that can foster tumor progression. This review summarizes the interactions between cancer cells, recruited immune cells and tissue-resident cell subpopulations, such as airway epithelial cells and alveolar macrophages, during homeostasis and disease. Furthermore, we examine the role of the lung immune landscape in response to current therapeutic interventions for cancer. Given the prevalence of lung malignancies, we propose that consideration of lung physiology as a whole is necessary to understand and treat these lethal diseases. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

转录因子Foxp3在肺癌细胞的表达及其意义

目的研究转录因子Foxp3在肺癌细胞中的表达及其对肿瘤免疫的影响。方法用常规RT-PCR检测Foxp3在肺癌细胞系中的表达,进一步用Western blot验证其蛋白水平的表达;将表达Foxp3的肿瘤细胞及用Foxp3 siRNA沉默后的肿瘤细胞分别与CD4+CD25-T细胞共培养,CFSE评价免疫效应细胞增殖情况。结果 Foxp3在肺癌细胞系中出现表达;肺癌细胞与CD4+T细胞进行共培养,可显著抑制共培养体系中CD4+T细胞增殖;肺癌细胞Foxp3表达沉默后,可显著改善共培养体系中肺癌细胞对CD4+T细胞增殖的抑制作用。结论在肺细胞癌细胞系发现Foxp3的表达,肺癌细胞Foxp3的表达参与对肿瘤特异性T细胞增殖的抑制。     

Association between COX-2 polymorphisms and non-small cell lung cancer susceptibility

Aim: To explore the association between COX-2 polymorphisms and non-small cell lung cancer (NSCLC) susceptibility. Methods: We collected fasting peripheral venous blood from 60 cases with NSCLC and 62 healthy controls through physical examinations, and applied PCR-RFLP to analyze COX-2 polymorphisms of two groups. Results: With respect to detecting COX-2 rs689466 and rs5275 polymorphisms, the distribution frequency of mutant genotype AA of COX-2 rs689466 in case group was higher than that in control group, which possessed significant difference between two groups (P < 0.05). Carriers with AA genotype were 4.05 times at risk of NSCLC than those with GG genotype (P = 0.04, OR=4.05, 95% CI = 1.14-14.43). The distribution of mutant genotype CC of COX-2 rs5275 was different between two groups, and carriers with genotype CC were at 5.70 times higher risk of NSCLC than those with genotype TT. After corrected by sex, gender, smoking and drinking factors, AA genotype of COX-2 rs689466 and CC genotype of COX-2 rs5275 still contributed to increased risk of NSCLC (OR=4.22, 95% CI=1.10-16.17, OR=6.95, 95% CI=1.27-38.11). After analyzed of linkage disequilibrium (LD) and haplotypes of alleles in two SNPs, the distribution frequency of A-C haplotype in case group was higher than that in control group, with significant difference between two groups (P < 0.05). After corrected by sex, gender, smoking and drinking factors, statistical difference was still found in the total distribution of A-C haplotype between two groups (P = 0.03, OR=6.11, 95% CI=1.16-32.2). Conclusions: COX-2 rs689466 and rs5275 polymorphisms may be related to NSCLC susceptibility. And A-C haplotype might be a susceptibility haplotype for NSCLC.     

NKG2D与NKG2DL在肺癌免疫调节中的作用及临床意义

NKG2D与NKG2DL是目前研究的热点,NKG2D可表达于几乎所有的NK细胞、CD8+αβT细胞、γδ T细胞及少量CD4+αβT细胞的细胞膜表面,与其配体NKG2DL结合后,可激活NK细胞及T细胞,诱导机体的抗肿瘤免疫应答,杀伤表达NKG2DL的肿瘤细胞,因此,NKG2D及其配体NKG2DL在肿瘤的免疫调节过程中起着极其重要的作用.目前肺癌的预后仍不容乐观,迫切需要发展一种新型的治疗方法来达到特异杀灭肿瘤细胞而不损伤或仅轻微损伤正常细胞的目的,这使得免疫疗法成为一种极具吸引力及应用前景的治疗肺癌的方法.     

Galectin-3的抗凋亡机制及其在肺癌中的研究

半乳凝素-3(Galectin-3,Gal-3)是糖结合蛋白家族中的一员,能与β-半乳糖苷特异性结合,参与许多生理和病理过程,在调解细胞生长与分化、机体免疫、细胞黏附、炎症反应、细胞凋亡、血管侵袭等诸多方面起着重要的作用,且与肿瘤的侵袭、转移及预后有关.目前,在许多恶性肿瘤中都发现了Galectin-3的表达,如胃癌、结肠癌、乳腺癌、膀胱癌、甲状腺癌.但在不同的肿瘤中,Galectin-3的表达水平不同.在一些肿瘤中,Galectin-3表达增高,而在某些肿瘤中其表达降低,且其亚细胞定位与肿瘤的发生、发展密切相关.     

Discovering novel lung cancer associated antigens and the utilization of their autoantibodies in detection of lung cancer

ObjectiveThe identification of tumor-associated antigens (TAAs) and their corresponding autoantibodies in lung cancer (LC) may expand our vision of cancer immunity. This study aims to screen novel TAAs to distinguish LC from the healthy population.MethodsIn our previous study, 35 genes encoding LC-associated TAAs were identified from the serological analysis of recombinant cDNA expression libraries (SEREX), and Oncomine database was further used to identify potential genes in cancer progression. Autoantibody to TAAs were tested by enzyme-linked immunosorbent assay (ELISA) in sera from 1379 participants in validation set and verification set.FindingsBased on analysis of three independent microarrays in Oncomine, ten genes were consistently dysregulated in LC. The sera level and positive frequency of the anti-TOP2A, anti-ACTR3, anti-RPS6KA5 and anti-PSIP1 from LC patients were higher than normal control in validation set. The area under curve (AUC) of anti-TOP2A, anti-ACTR3, anti-RPS6KA5 and anti-PSIP1 was respectively 0.758, 0.787, 0.707, 0.668. The sensitivity of these four autoantibodies for LC detection ranged from 26.63 % to 32.07 % with the specificity over 90 %. Data from the verification set confirmed the results. Except that, the frequency of serum autoantibody against TOP2A (43.3 %) and ACTR3 (50.0 %) was significantly higher in early stage LC than late stage (23.6 % and 22.3 %, respectively).ConclusionTOP2A, ACTR3, RPS6KA5 and PSIP1 can elicit humoral immune response in LC and their autoantibodies have relationship with the tumorigenesis of LC. Anti-TOP2A and anti-ACTR3 have the potential to serve as a serological biomarkers in early stage LC.