非小细胞肺癌分泌蛋白的蛋白质组学研究

背景与目的:癌细胞分泌蛋白是潜在的血清标志物,而且对肿瘤的早期筛选也具有重要意义。因此,系统、全面地研究肿瘤细胞分泌蛋白对于肿瘤血清标志物的筛选具有重要意义。本研究旨在鉴定肺癌细胞分泌蛋白,为肺癌血清标志物筛选提供实验依据。方法:用含胎牛血清的RPMI-1640培养液培养非小细胞肺癌细胞系A549细胞,收集A549细胞培养液中的蛋白及胎牛血清培养液中的蛋白(对照)进行蛋白双向电泳,应用基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)及生物信息学鉴定A549凝胶上特有的蛋白质点。筛选出人属的蛋白质点后,应用RT-PCR方法检测其在肺癌组织及配对远处肺组织中的表达差异;并检测培养液及血清中锰-超氧化物歧化酶(manganesesuperoxidedismutase,Mn-SOD)水平。结果:鉴定出人属的蛋白质点14个,包括肌动蛋白、!烯醇酶(alphaenolase,ENO1)、Mn-SOD、谷胱苷肽S转移酶P(glutathioneS-transferaseP,GSTP1-1)、二氢二醇脱氢酶(dihydrodioldehydrogenase,DDH)、磷酸甘油酸变位酶1(phosphoglyceratemutase1,PGAM1)、葡萄糖依赖性胰岛素释放肽受体(glucose-dependentinsulinotropicproteinreceptor,GIPR)、肽基脯氨基顺反异构酶(peptidyl-prolylcis-transisomeraseA,PPIA)、磷脂酰乙醇胺结合蛋白(phosphatidylethanolaminebindingprotein,PEBP)、蛋白基因产物9.5(ubiquitincarboxyl-terminalhydrolaseisozymeL1,PGP9.5)、peroxiredoxin1(PDX1)、galectin-1及专利蛋白WO0222660等。RT-PCR检测示ENO1、DDH、PEBP、PGP9.5、PDX1、PGAM1、PPIA在肺癌组织中高表达。酶活性检测发现A549培养液中存在Mn-SOD,且发现非小细胞肺癌患者血清中有高水平的Mn-SOD。结论:本研究首次对非小细胞肺癌细胞的分泌蛋白进行了鉴定;发现多种高表达的非小细胞肺癌分泌蛋白基因,其中首次发现ENO1及PEBP基因在非小细胞肺癌中高表达;Mn-SOD是非小细胞肺癌的分泌型血清标志物。该研究为非小细胞肺癌血清标志物的筛选提供了新的方法和候选分子。     

分泌蛋白二氢二醇脱氢酶的蛋白组学鉴定及其在非小细胞肺癌中的研究

目的：鉴定非小细胞肺癌（non—small cell lung cancer，NSCLC）细胞的分泌蛋白，以期为肺癌血清标志物的筛选提供候选分子。方法：培养NSCLC的A549细胞系细胞，收集其培养液中的蛋白，应用蛋白质组学和质谱分析方法鉴定。鉴定的蛋白采用Western印迹进一步验证。收集15例NSCLC癌组织及远处正常肺组织，采用RT—PCR、Western印迹法及免疫组织化学方法检测其在癌组织及肺组织中的表达差异及表达部位。收集血清104例，其中NSCLC患者64例、肺部良性肿瘤20例，健康人20例，应用ELISA检测血清中该分泌蛋白的变化。结果：在A549细胞的培养液中鉴定并证实存在二氢二醇脱氢酶（dihydrodiol dehydrogenase，DDH）。RT—PCR、Western印迹法及免疫组化检测发现：与正常肺组织比较，癌组织中有DDH mRNA和蛋白的高表达（P〈0．05），胞质和胞膜中均有DDH蛋白的丰富表达。血清ELISA检测提示，NSCLC患者血清中DDH浓度较肺部良性肿瘤患者及健康人高（P〈0．05）。结论：DDH在肺癌组织中高表达，且非小细胞肺癌患者血清中浓度升高，是非小细胞肺癌的分泌蛋白型血清标志物。     

非小细胞肺癌细胞分泌蛋白谱的鉴定

目的筛选非小细胞肺癌潜在的早期诊断标志物，对非小细胞肺癌细胞系A549的分泌蛋白进行鉴定。方法收集A549细胞培养上清中的蛋白进行蛋白双向电泳，应用基质辅助激光解析电离飞行时间质谱（MALDI—TOF—MS）及生物信息学鉴定A549凝胶上特有的蛋白质点。结果鉴定出人属的蛋白质点17个，包括α肌动蛋白，γ肌动蛋白，α烯醇酶（ENO1），锰-超氧化物歧化酶（MnSOD），谷胱甘肽S转移酶P（GSTP1—1），Dihydrodiol dehydrogenase 2（DDH），磷酸甘油酸变位酶1（PGAM1），葡萄糖依赖性胰岛素释放肽受体（GIPR），肽基脯氨基顺反异构酶（PPIA），磷脂酰乙醇胺结合蛋白（PEBP），蛋白基因产物9．5（PGP9．5），Peroxiredoxin1（PDX1），Galectin-1及专利蛋白WO0222660等。结论该研究为非小细胞肺癌早期诊断标志物的筛选提供了新的方法和候选分子。     

血清GST-π在非小细胞肺癌诊断中的应用价值

目的 :测定肺癌患者血清 GST- π表达 ,探讨其作为非小细胞肺癌肿瘤标志物的临床应用价值。方法 :应用双抗夹心 EL ISA法检测 114例非小细胞肺癌、8例小细胞肺癌、11例肺良性病变及 6 0例健康对照者血清 GST- π含量。结果 :非小细胞肺癌患者血清 GST- π含量、阳性率明显高于健康对照者、小细胞肺癌和良性病变患者 ,血清 GST- π与非小细胞肺癌 TNM分期无关 ,与分化程度有相关性。结论 :血清 GST- π含量可作为非小细胞肺癌的肿瘤标志物 ,血清 GST- π含量测定对非小细胞肺癌的临床诊断有一定应用价值     

分泌型簇集素蛋白在肺癌细胞株中生物学行为的研究

背景与目的：簇集素蛋白(clusterin,CLU)在肺癌的发生、发展中发挥重要作用。本研究用分泌型CLU体外处理肺癌细胞株,旨在探讨分泌型CLU对肺癌细胞株的影响。方法：用真核表达纯化的分泌型CLU处理非小细胞肺癌细胞株H460、A549,采用Boyden小室试验检测细胞的迁移能力,用CCK8检测细胞的增殖能力,利用芯片技术分析CLU处理H460细胞的microRNA表达谱,然后采用实时荧光定量PCR检测分泌型CLU处理H460细胞中microRNA的表达,初步分析分泌型CLU执行肿瘤生物学功能的microRNA相关分子机制。结果：分泌型CLU处理组H460迁移的细胞是BSA处理组的3.10倍(P<0.0001);分泌型CLU处理组A549迁移的细胞是BSA处理组的3.40倍(P<0.0001);分泌型CLU抑制了肺癌细胞株H460/A549的生长(P<0.0001);利用芯片技术发现microRNA-302b-3p、microRNA-23a-5p和microRNA-101-5p高表达。结论：在非小细胞肺癌细胞株H460/A549中,真核表达的分泌型CLU促进了细胞迁移、抑制了细胞生长,并且能引起microRNA表达谱的改变,本研究为探索分泌型CLU在非小细胞肺癌中的作用提供了重要的实验依据。     

人脐带间充质干细胞及其条件培养液对非小细胞肺癌多倍体A549细胞增殖、迁移和凋亡的影响

目的:探讨人脐带间充质干细胞（hUC-MSC）及其条件培养液对人非小细胞肺癌多倍体A549细胞增殖、迁移和凋亡的影响。方法:取对数生长期的A549细胞,采用1 μmol/L多西他赛诱导24 h后,更换为含10%胎牛血清的DMEM/F12培养液继续培养3 d,建立多倍体A549细胞模型。分离与培养hUC-MSC,制备hU...     

CTEN通过TGF-β1促进非小细胞肺癌细胞紫杉醇耐药的作用及机制研究

目的:探讨CTEN通过TGF-β1促进非小细胞肺癌（NSCLC）细胞紫杉醇耐药的作用和分子机制。方法:采用逐步增加剂量间歇作用的方法诱导非小细胞肺癌紫杉醇耐药细胞系并命名为A549/R,采用定量PCR和Western blotting检测耐药细胞和亲本细胞中CTEN和TGF-β1 mRNA及蛋白的表达,应用Lipofectamine 2000分别将CTEN高表达质粒pcmv-CTEN及其对照质粒pcmv转染至非小细胞肺癌A549细胞,分别为高表达组和对照组,分别用定量PCR和Western blotting检测CTEN和TGF-β1 mRNA及蛋白水平的变化;MTT法检测A549细胞对紫杉醇的敏感性及细胞增殖。应用Lipofectamine 2000分别将CTEN干扰质粒siCTEN及其对照siNC转染至紫杉醇耐药细胞系A549/R细胞,分别为干扰组和对照组,分别用定量PCR和Western blotting检测CTEN和TGF-β1 mRNA及蛋白水平的变化;MTT法检测A549/R细胞对紫杉醇的敏感性及细胞增殖。应用Lipofectamine 2000分别将TGF-β1干扰质粒siTGF-β1及其对照质粒siNC转染至非小细胞肺癌A549细胞,分别为干扰组和对照组,分别检测两组细胞中TGF-β1 mRNA及蛋白水平的表达情况,然后再分别用Lipofectamine 2000将CTEN高表达质粒pcmv-CTEN转染入两组细胞,MTT法检测A549细胞对紫杉醇的敏感性及细胞增殖。结果:成功构建在5 μg/mL紫杉醇中稳定生长的非小细胞肺癌耐药细胞系A549/R。定量PCR和Western blotting显示,CTEN和TGF-β1 mRNA及蛋白在紫杉醇耐药细胞系A549/R中的表达明显高于在其亲本细胞系A549中的表达;与对照组相比,高表达CTEN组的A549细胞中TGF-β1表达上升,细胞对紫杉醇的敏感性降低,细胞增殖增强;与对照组相比,低表达CTEN组的A549/R细胞中TGF-β1表达降低,细胞对紫杉醇的敏感性升高,细胞增殖减弱;在A549细胞中低表达TGF-β1后再过表达CTEN,其促进紫杉醇耐药及细胞增殖的作用也明显减弱。结论:CTEN具有促进非小细胞肺癌A549的紫杉醇耐药,促进细胞增殖的作用,其发生机制可能与上调TGF-β1的表达有关。     

NSCLC患者血清中HSP90α的表达及其临床意义

目的:探讨热休克蛋白90α（HSP90α）在非小细胞肺癌（NSCLC）患者血清中的表达水平及其临床意义。方法:选取2015年10月至2016年10月期间，在第四军医大学唐都医院胸外科确诊为NSCLC的患者100例为肺癌组。选取同期在我院进行体检的健康成年人40例为对照组。检测并比较两组血清HSP90α表达水平差异；比较不同肿瘤分期(TNM分期)患者的血清HSP90α水平差异；探讨不同TNM分期与血清HSP90α表达水平之间的关系。培养NSCLC A549细胞，检测其分泌至胞外的HSP90α蛋白；使用HSP90α抑制剂处理A549细胞，MTT法检测细胞增殖能力的变化。结果:肺癌组与对照组患者血清HSP90α表达水平比较，差异有统计学意义（P＜0.001）；不同TNM分期患者血清HSP90α表达水平比较，差异有统计学意义（P<0.001）；Spearman秩相关分析显示，TNM分期与血清HSP90α水平呈正相关(r=0.95，P＜0.05)。体外细胞实验证实A549细胞能分泌HSP90α至细胞外，使用HSP90α抑制剂处理后细胞增殖能力明显下降。结论:NSCLC患者血清中HSP90α表达水平较正常人升高，并且与TNM分期呈正相关。血清HSP90α由肺癌细胞分泌入血，检测其水平对肺癌的诊断与治疗有一定的临床参考价值。     

肺癌细胞自分泌VEGF对mCRPs的调控及其机制

目的：研究人非小细胞肺癌A549细胞自分泌VEGF对膜结合补体调节蛋白(membranebounal complement regulatory proteins, mCRPs)的调控及其机制。方法: RTPCR法检测CD46、CD55、CD59、VEGF及其受体(KDR和FLT1)和 IL8及其受体(CXCR1 CXCR2) mRNA在人非小细胞肺癌A549细胞的表达。MTT法检测抗VEGF抗体和抗IL8抗体对A549细胞增殖的影响,流式细胞术检测抗VEGF抗体和抗IL8抗体对A549细胞mCRPs表达水平的影响,Western blotting检测抗VEGF抗体对转录因子KLF2和磷酸化NFκB p65蛋白表达的影响。结果：A549细胞表达膜结合型CD46、CD55和CD59 mRNA,亦表达VEGF及其受体（KDR和FLT1）和 IL8及其受体（CXCR1和CXCR2） mRNA。抗VEGF抗体明显抑制A549细胞的增殖（P<0.05）。终质量浓度0.1 μg/ml抗VEGF抗体封闭72 h,CD55和CD59 mRNA表达下降,膜结合的CD55和CD59蛋白分子表达降低(均P<0.05);胞质和核KLF2蛋白相对定量值分别从063和0.88下降至0.42和0.66,胞质和胞核磷酸化NFκB p65蛋白相对定量值分别从0.44和0.28下降至0.37和019。结论：A549细胞可能通过自分泌VEGF增加NFκB p65和KLF2转录因子水平,从而上调CD55和CD59的表达。     

人附睾蛋白4在非小细胞肺癌组织及血清中的表达及临床意义

目的 研究人附睾分泌蛋白4(HE4)在非小细胞肺癌(NSCLC)组织及血清中的表达及临床意义.方法 收集124例NSCLC患者(NSCLC组)的血液标本和肺癌组织标本,其中行手术治疗的患者取癌旁组织和正常肺组织,并在术后3天再次抽取血液标本;同期收集96例健康体检者(健康组)的血清标本.采用电化学发光免疫分析仪检测血清中HE4浓度;免疫组化EnVision二步法检测非小细胞肺癌组织、癌旁组织、正常肺组织中HE4的表达.结果 HE4在肺癌组织、肺腺癌组织、Ⅲ~Ⅳ期肺癌组织中的阳性表达率分别高于癌旁组织和正常肺组织、肺鳞癌组织、Ⅰ~Ⅱ期肺癌组织(P<0.05);NSCLC组患者的血清HE4浓度高于健康体检组(P<0.05);手术治疗的患者术后3天血清HE4浓度低于术前血清HE4浓度(P<0.05);NSCLC组患者血清HE4水平与病理分期、组织中HE4阳性表达呈正相关,与年龄、性别、病理类型无明显相关性.结论 NSCLC组织及血清HE4表达升高,可作为NSCLC的肿瘤标志物,是NSLCL的早期辅助诊断、疗效评价、预后评估的客观指标,可用于指导临床的综合治疗.     

Proteomics-based identification of secreted protein dihydrodiol dehydrogenase as a novel serum markers of non-small cell lung cancer

Identification of secreted proteins of lung cancer could provide new candidates of serum biomarkers for cancer diagnosis and prognosis evaluation. In this study, non-small cell lung cancer (NSCLC) cell line A549 was cultured. Proteins in the conditioned medium of A549 were recovered and the proteome analysis was subsequently performed. Secreted proteins of A549 were identified using mass spectrometry and database search. Fourteen human proteins were identified, including peptidyl-prolyl cis-trans isomerase A, manganese superoxide dismutase, peroxiredoxin 1, phosphatidylethanolamine-binding protein, glutathione S-transferase P, PGP9.5, alpha enolase, phosphoglycerate mutase 1, galectin-1 and dihydrodiol dehydrogenase (DDH). DDH was selected for further analysis using RT-PCR, immunoblotting, immunohistochemical staining and ELISA in NSCLC patients. Compared with normal lung tissues, higher DDH mRNA and protein expression level were found in 15 NSCLC cancer tissues (p<0.05). DDH overexpression was identified to be located in cytoplasm and cell membrane by immunohistochemical staining in NSCLC tissue. The serum level of DDH was significantly higher in NSCLC patients (n=64) than nonmalignant lung tumor (n=20) and healthy controls (n=20) (p<0.05). The results show that DDH was one of the secreted proteins in NSCLC. It can serve as a tissue marker and a novel serological marker of NSCLC. Identification of secreted proteins could be a feasible and effective strategy to search potential serum biomarkers of cancer.     

Production of immunoreactive insulin-like growth factor-I (IGF-I) and IGF-I binding proteins by human lung tumours.

The production of insulin-like growth factor I (IGF-I) and IGF-I binding proteins (BPs) by human lung tumour cell lines in vitro has been examined and the levels of these substances in the serum of lung cancer patients investigated. While small cell lung cancer (SCLC) cell lines secreted both IGF-I and BPs, non-small cell lung cancer (NSCLC) cell lines secreted BPs only. No evidence of increased serum IGF-I levels was obtained in a cohort of 52 lung cancer patients having SCLC and NSCLC histologies. In contrast, serum levels of low molecular weight BPs were markedly elevated in the majority of lung cancer patients.     

Recoverin as a paraneoplastic antigen in lung cancer: the occurrence of anti-recoverin autoantibodies in sera and recoverin in tumors

Using immunoblotting with recombinant recoverin as an antigen, we have examined 279 serum samples from individuals with small cell lung carcinoma (SCLC, 99 patients), non-small cell lung carcinoma (NSCLC, 44 patients), and non-malignant pulmonary disorders (86 patients) as well as sera from 50 healthy donors. Autoantibodies against recoverin (anti-Rc) were detected in sera from 15 patients with SCLC (15% of cases) and from 9 patients with NSCLC (about 20% of cases). Only two anti-Rc positive cases were detected in patients with non-malignant pulmonary disorders, while no such cases were found in healthy individuals. Immunohistochemical investigation of paraffin sections of 44 SCLC and 40 NSCLC tumors revealed recoverin-positive reaction in 30 SCLC (68%) and 34 NSCLC (85%) sections. Despite the high specificity (98%), the low sensitivity (less than 20%) does not allow serum anti-Rc to be considered as a valuable marker of lung cancer. However, taking into account the high occurrence of aberrant expression of recoverin in lung tumors, this PNA could be considered as a potential target for immunotherapy of lung cancer.     

VIP增强VEGF mRNA在非小细胞肺癌细胞中的表达

背景与目的研究发现血管内皮生长因子(VEGF)和血管活性肠肽(VIP)均对肿瘤的生长具有促进作用,但VIP通过何种方式来促进肿瘤的生长还不清楚。本研究的目的是观察VIP对非小细胞肺癌(NSCLC)细胞中VEGFmRNA表达的影响。方法应用反转录聚合酶链式反应(RTPCR)技术检测VEGFmRNA在NSCLC细胞系和小细胞肺癌(SCLC)细胞系中的表达及VIP对其表达的影响。结果在NSCLC细胞系A549、GLC82、H157、H460和SCLC细胞系H446中检测到了VEGFmRNA的表达。VIP能促进VEGFmRNA在A549和H157细胞中的表达,在VIP作用8h和16h时,VEGFmRNA的表达水平达到最高,显著高于VIP作用0h时(P<0.01)。结论VIP可能通过增强VEGFmRNA在肺癌细胞中的表达和分泌,促进肺癌新生血管的生成,参与肿瘤的生长。     

Proteomics-based identification of protein gene product 9.5 as a tumor antigen that induces a humoral immune response in lung cancer.

We used a proteomic approach to identify proteins that commonly induce an antibody response in lung cancer. Sera from 64 newly diagnosed patients with lung cancer, 99 patients with other types of cancer, and 71 noncancer controls were analyzed for antibody-based reactivity against lung adenocarcinoma proteins resolved by two-dimensional PAGE. Unlike controls, autoantibodies against a protein identified by mass spectrometry as protein gene product 9.5 (PGP 9.5) were detected in sera from 9 of 64 patients with lung cancer. Circulating PGP 9.5 antigen was detected in sera from two additional patients with lung cancer, without detectable PGP 9.5 autoantibodies. PGP 9.5 is a neurospecific polypeptide previously proposed as a marker for non-small cell lung cancer, based on its expression in tumor tissue. Using A549 lung adenocarcinoma cell line, we have demonstrated that PGP 9.5 was present at the cell surface, as well as secreted. Thus, the findings of PGP 9.5 antigen and/or antibodies in serum of patients with lung cancer suggest that PGP 9.5 may have utility in lung cancer screening and diagnosis.     

Characterization and Purification of an Immunosuppressive Factor Produced by a Small Cell Lung Cancer Cell Line

The present study was undertaken to determine whether small cell lung cancer (SCLC) cell lines produce immunosuppressive factors and, if they do, to characterize the factors. The supernatants of SCLG cell lines, H69 and N857, inhibited not only the blastogenic response of human peripheral blood lymphocytes (PBL) to phytohemagglutinin or concanavalin A, but also the cytotoxic activity of lymphokine-activated killer cells. Neither was inhibited by supernatants from non-SCLC cell lines PC9, QG56, and A549. The immunosuppressive activity of H69 supernatant was stable upon heating to 56°C for 60 min, but labile when heated to 70°C for 10 min. The activity was abolished after dialysis at pH 2.0 or pH 11.0, but not at pH 4.5 or pH 9.0. Digestion with trypsin or proteinase eliminated the immunosuppressive activity, whereas treatment with neuraminidase, mixed glycosidase, DNase or RNase had no effect, suggesting that the immunosuppressive activity in H69 supernatant is due to a protein factor. This H69-derived immunosuppressive factor was isolated by ion exchange chromatography using a gradient of 0.04 to 0.08 M NaCl solution. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the factor to have molecular weights of 98 kD and 102 kD, respectively. These results suggest that SCLC cells produce a potent immunosuppressive factor which may account for the immune deficiency in SCLC patients.     

Detection of circulating adenocarcinoma-associated antigen in the sera of cancer patients with a monoclonal antibody

The antigen (YH206 antigen) corresponding to the monoclonal antibody (MoAb) YH206 (IgM), which was produced against a lung adenocarcinoma cell line A549, was found to be an extremely high-molecular-weight protein of over 330,000 daltons by means of SDS-PAGE and Western blot analysis. The immunohistological distribution of the antigenic determinant recognized by MoAb YH206 was found to be limited to adenocarcinoma tissues. It was shown by reversed passive hemagglutination assay (RPHA) that the antigen could be detected not only in the spent medium from human lung cancer cell line A549 but also in the sera from cancer patients. Only 3 out of 30 (10%) healthy donors and 4 of 31 (12.9%) patients with benign diseases had serum antigen levels of more than 1/64 dilution. In contrast, 42 of 87 (48.3%) patients with lung cancer and 29 of 50 (58.0%) patients with cancers of the digestive organs had elevated levels of the antigen. As regards the relation between antigen levels and clinical stages of lung cancer, values of more than 1/128 dilution were detected only in patients with stage III or IV disease.