非小细胞肺癌T7噬菌体展示文库的构建

背景与目的SEREX(Serological analysis of recombinant expression libraries)是近年来发展起来的一种寻找肿瘤相关抗原的方法,通过与噬菌体展示技术结合,可以有效和快速的筛选肿瘤相关抗原。本研究拟构建人肺鳞癌和肺腺癌T7噬菌体展示cDNA文库,为进一步筛选和鉴定非小细胞肺癌相关抗原打下基础。方法诊断明确的肺鳞癌和腺癌患者手术标本各5例,Trizol试剂提取总RNA,Straight A's系统分离鳞癌和腺癌mRNA,逆转录合成双链cDNA后,与T7Select 10-3载体连接,体外包装并扩增得到人肺鳞癌和腺癌T7噬菌体展示cDNA文库。结果构建肺鳞癌和肺腺癌T7噬菌体展示cDNA文库各一个,原始文库重组克隆数分别为鳞癌1.8×106pfu,腺癌5×106pfu。扩增后滴度为3.2×1010pfu/mL和2.5×1010pfu/mL。随机从2个文库中各抽取24个克隆PCR扩增插入片断,电泳结果显示肺腺癌文库所有克隆均有插入片段,肺鳞癌文库插入率超过95%(23/24)。两个文库插入片断大小均在(300-1500)bp之间。结论成功构建了人肺鳞癌和腺癌T7噬菌体展示cDNA文库,为下一步筛选和鉴定非小细胞肺癌相关基因奠定基础。     

非小细胞肺癌T7噬菌体展示文库的构建

Objective: Currently, only a limited numbers of tumor markers for non small lung cancer (NSCLC) diagnosis, new biomarker, such as serum autoantibodies may improve the early detection of lung cancer. Our objective is construction human lung squamous carcinoma and adenocercinoma T7 phage display cDNA library from the tissues of NSCLC patients. Methods: mRNA was isolated from a pool of total RNA extract from NSCLC tissues obtained from 5 adenocarcinomas and 5 squamous carcinomas, and then mRNA was reverse transcribed into double stranded cDNA. After digestion, the cDNA was inserted into T7Select 10-3 vector. The phage display cDNA library was constructed by package reaction in vitro and plate proliferation. Plaque assay and PCR were used to evaluate the library. Results: Two T7 phage display cDNA library were established. Plaque assay show the titer of lung squamas carcinoma library was 1.8×106 pfu, and the adenocarcinoma library was 5×106pfu. The phage titer of the amplified library were 3.2×1010 pfu/mL and 2.5 x 1010 pfu/mL. PCR amplifica-tion of random plaque show insert ratio were 100% (24/24) in adenocarcinorna library and 95.8% in human lung squamas carcinoma library (23/24). Insert range from 300 bp to 1 500 bp. Conclusion: Two phage display cDNA library from NSCLC were constructed.     

肾癌T7噬菌体展示肽库构建的实验研究

目的:构建肾细胞癌T7噬菌体展示肽库,为下一步筛选肾癌早期诊断分子标志群打下基础.方法:用传统Trizol方法分别抽取31例涵盖各种组织类型肾癌组织标本的总RNA,确定完整性后再根据OD值等量混合总RNA,用试剂盒完成mRNA的分离并电泳检测其质量,经反转录、末端平齐、片段长短筛选、加接头、双酶切、去除多余接头和<300 bp的cDNA片段等步骤,再与T7Select10-3b载体连接、体外包装并扩增得到肾癌T7噬菌体展示cDNA文库.通过铺平板测定和PCR技术鉴定所建文库质量.结果:建成原始文库的库容量为5.0×107 pfu/mL,扩增后文库滴度为3.5×1012 pfu/mL,重组率为95%,插入片段为300～2 000 bp.结论:成功用T7噬菌体构建了高质量的肾癌cDNA文库,为筛选可用于临床早期诊断的肾癌特异标志奠定基础.     

KDR基因重组的T7噬菌体疫苗构建及其对小鼠Lewis肺癌抑制作用的研究

目的：构建KDR基因重组的T7噬菌体疫苗以及探讨其对小鼠Lewis肺癌的抗肿瘤作用。方法：克隆KDR的膜外Ⅱ区基因,构建KDR基因重组T7噬菌体疫苗,免疫小鼠,免疫四次后接种Lewis肺癌,观察肿瘤的生长情况,14 d后脱颈椎杀死小鼠,眼球取血、取出瘤体称重,计算抑瘤率,观察抗肿瘤效果。ELISA检测小鼠血清抗KDR抗体滴度。结果：实验组抑制程度明显,肿瘤生长速度最慢,肿瘤重量与生理盐水组和空白噬菌体组相比有统计学差异(P＜0．05),抑瘤率可达57．0%,远大于空白噬菌体组(16．0%)。小鼠血清稀释至1∶500,小鼠特异性抗人源KDR抗体仍呈阳性。结论：KDR基因重组的T7噬菌体疫苗对小鼠Lewis肺癌具有明显的抗肿瘤作用,用人源KDR作为免疫原免疫小鼠通过免疫交叉反应可以打破对自身抗原的免疫耐受,产生特异性的免疫反应。     

肺癌患者手术前后NK细胞及T细胞亚群的变化

 本文通过对 5 6例肺癌患者手术前后外周血ＮＫ细胞活性、Ｔ细胞亚群的动态观察 ,探讨外科手术对肺部患者细胞免疫功能的影响。1　资料与方法1.1　研究资料　肺癌 5 6例 ,男 4 5例 ,女 11例 ,年龄 2 3～ 75岁 ,平均年龄 6 1.5岁。肺癌根治术者 2 0例 ,非根治术者36例。全部患者均为来我院的初治患者 ,并经手术后病理学检查确诊 ,术前 1周内体温、血象正常 ,肝肾功能正常 ,未用影响免疫功能的药物。 4 6例健康人血标本 ,来自我院体检的正常志愿者。1.2　方法1.2 .1　ＮＫ细胞活性检测采用ＳＡＰ法 ,Ｔ淋巴细胞亚群检测采用ＳＰ法 ,上述试剂盒均由北京中山生物技术有限公司提供。1.2 .2　所有肺癌病例均于术前 1周内 ,术后 2 1天空腹抽取用肝素抗凝的外周血 ,进行ＮＫ细胞及Ｔ细胞亚群的检测。1.2 .3　统计学处理　所有数据资料均使用ＰＥＭＳ软件进行方差分析及ｔ检验。2　结果　　术前及术后 2 1天根治组与非根治组ＮＫ细胞活性及Ｔ细胞亚群变化。　　肺癌患者细胞免疫功能低下 ,其ＮＫ细胞活性及ＣＤ3 + 、ＣＤ4+ 明显低于对照组 ,而ＣＤ8+ 则显著增加 ,致使ＣＤ4+ /Ｃ...     

化疗对晚期肺癌患者T淋巴细胞亚群和红细胞免疫功能的影响

目的：分析42例晚期肺癌者化疗前后T淋巴细胞亚群和红细胞免疫功能的变化,并探讨其与病情的关系。方法：对42例晚期肺癌化疗前后的血标本采用流式细胞术检测T淋巴细胞亚群和采用交体粘附法检测红细胞免疫功能,并与体检健康者作比较,结果：本组晚期肺癌患者治疗前后T淋巴细胞亚群和红细胞免疫功能均低于对照组（P＜0．05,P＜0．01）。治疗后,化疗有效率总T细胞（CD3^ ）、辅助／诱导T淋巴（CD4^ ）、CD4^ 与CD8^ 比值均显著升高（P＜0．01）,细胞毒／抑制性T淋巴细胞（CD8^ ）降低（P＜0．05）;化疗无效者CD3^ 、CD4^ 、CD4^ ／CD8^ 显著降低（P＜0．05,P＜0．01）,而CD8^ 升高（P＜0．01）,直向肿瘤红细胞花环（direct tumor erythrocyte rosette,DTER）,红细胞C3b受体花环（red blood cell C3b receptor rosette,RBC-C3bRR)和红细胞免疫复合物花环（red blood cell immunity complex rosette,RBC-ICR),化疗前后变化不明显（P＞0．05）。结论：晚期肺癌患者免疫功能低下,有铲化疗能提高患者的T淋巴细胞亚群免疫功能,临床上对免疫功能的观察对肺癌患者的治疗和预后有一定的监测作用。     

人源性肺癌噬菌体展示单链抗体库的构建

目的:构建人源性肺癌噬菌体单链抗体库,为筛选肺癌相关抗原的抗体奠定基础。方法:提取肺癌转移淋巴结总RNA,用RT-PCR技术扩增人抗体重链可变区(VH)和轻链可变区(VL)基因,在体外将VH和VL连接成单链抗体(ScFv)基因,并克隆到噬菌粒载体pCANTAB 5E中,电转化至感受态的大肠杆菌TG1,经辅助噬菌体超感染,形成噬菌体单链抗体库,采用限制性内切酶鉴定其多样性。结果:从肺癌转移淋巴结中成功提取RNA,逆转录PCR扩增出人可变区基因,连接形成单链抗体,最终构建了库容为1.2×108的抗人肺癌单链抗体库。BstNⅠ酶切法证明构建的抗体库具有良好的多样性。结论:成功地构建了噬菌体展示的抗人肺癌单链抗体库,为进一步筛选肺癌相关蛋白的可溶性抗体奠定了基础。     

胃癌组织中T细胞及其亚群ICOS分子的表达及其意义

目的探讨胃癌组织中T细胞共刺激分子ICOS及其亚群的表达及意义。方法应用流式细胞术检测38例胃癌和21例健康人（对照组）外周血T细胞亚群及其共刺激分子ICOS的表达。结果胃癌组与对照组比较：CD3^＋T细胞表达（53．61±13．84）／（72．07±7．83）％,P〈0．01;CD3^＋CD4^＋T细胞表达（29．84±9．71）／（38．79±5．08）％。P〈0．01;CD3^＋ICOS^＋T细胞表达（25．80±10．56）／（0．82±0．98）％,P〈0．01;CD3^＋CD8^＋ICOS^＋T细胞表达（1．57±1．99）／（0．02±0．04）％,P〈0．01;CD3^＋CD8^＋ICOS^-T细胞表达（16．06±6．94）／（20．56±6．54）％,P〈0．05。胃癌组患者手术前和手术后1周外周血T细胞亚群的差异无统计学意义（P〉0．05）。结论胃癌患者T细胞数量明显减少,T细胞共刺激分子ICOS表达增高,CD4^＋T细胞显著减少。     

肺癌患者化疗前后T细胞抑制性分子的研究

  目的  研究晚期肺癌患者化疗5个周期T淋巴细胞表面抑制性分子（TIM3、PD-1、CTLA-4）的变化。  方法  应用流式细胞仪技术检测33例初治晚期肺癌患者化疗5个周期T淋巴细胞表面抑制性分子的表达变化。并与23例健康志愿者作对比。  结果  化疗前晚期肺癌患者外周血T淋巴细胞表面抑制性分子的表达明显高于健康对照组, 差异具有统计学意义（P < 0.05）。化疗后19例临床疗效评价为PR或SD的患者, 其外周血CD4+TIM3+T淋巴细胞、CD8+TIM3+T淋巴细胞、CD4+PD-1+T淋巴细胞、CD8+PD-1+ T淋巴细胞比例在化疗5个周期中降低, 差异有统计学意义（P < 0.05）; 而CD4+CTLA-4+T淋巴细胞、CD8+CTLA-4+T淋巴细胞比例呈现下降趋势, 与化疗前相比无显著性差异（P>0.05）。  结论  晚期肺癌患者的免疫功能处于抑制状态。有效的化疗可降低T淋巴细胞表面抑制性分子表达, 改善肿瘤对免疫功能的抑制。      

肺癌射频消融术后CD4＋ T细胞亚群改变研究进展

CD4＋T细胞包括Th1、Th2、Th17及Treg细胞,它们在肿瘤免疫中发挥重要作用。肺癌射频消融（radiofrequency ablation,RFA）术后可使Th1细胞升高、Th2细胞降低、Th1/Th2细胞免疫失衡得以纠正,Th17及Treg细胞降至正常水平。CD4＋T细胞亚群的改变可作为肺癌RFA术后疗效评估和预后判断的指标。     

Screening and Identification of Peptides Specifically Targeted to Gastric Cancer Cells from a Phage Display Peptide Library

Background: Gastric cancer is the second most common cancer among the malign cancer types. Inefficiency oftraditional techniques both in diagnosis and therapy of the disease makes the development of alternative and noveltechniques indispensable. As an alternative to traditional methods, tumor specific targeting small peptides can be usedto increase the efficiency of the treatment and reduce the side effects related to traditional techniques. The aim of thisstudy is screening and identification of individual peptides specifically targeted to human gastric cancer cells usinga phage-displayed peptide library and designing specific peptide sequences by using experimentally-eluted peptidesequences. Methods: Here, MKN-45 human gastric cancer cells and HFE-145 human normal gastric epithelial cellswere used as the target and control cells, respectively. 5 rounds of biopannning with a phage display 12-peptide librarywere applied following subtraction biopanning with HFE-145 control cells. The selected phage clones were establishedby enzyme-linked immunosorbent assay and immunofluorescence detection. We first obtain random phage clonesafter five biopanning rounds, determine the binding levels of each individual clone. Then, we analyze the frequenciesof each amino acid in best binding clones to determine positively overexpressed amino acids for designing novelpeptide sequences. Results: DE532 (VETSQYFRGTLS) phage clone was screened positive, showing specific bindingon MKN-45 gastric cancer cells. DE-Obs (HNDLFPSWYHNY) peptide, which was designed by using amino acidfrequencies of experimentally selected peptides in the 5th round of biopanning, showed specific binding in MKN-45cells. Conclusion: Selection and characterization of individual clones may give us specifically binding peptides, butmore importantly, data extracted from eluted phage clones may be used to design theoretical peptides with betterbinding properties than even experimentally selected ones. Both peptides, experimental and designed, may be potentialcandidates to be developed as useful diagnostic or therapeutic ligand molecules in gastric cancer research.     

肺癌早期诊断相关标志物研究进展

肺癌是死亡率居于我国首位的肿瘤,早发现、早诊断是影响肺癌治疗效果的关键,但目前仍然缺乏精准安全的肺癌早期诊断方法,这极大地影响了肺癌患者的生存期。肺癌作为一种高度恶性肿瘤,其诊断生物标志物的相关研究受到了极大的关注。这些诊断生物标志物具有多样性,在肺癌早期诊断中的价值有待进一步探讨。     

血清肿瘤标记物、相关抗原及联合检测在肺癌患者诊断中的意义

目的 探讨血清肿瘤标记物、相关抗原的检测及联合检测在肺癌诊断中的实际意义.方法 随机选取98例肺癌患者(肺癌组)和同时期的68例肺良性病变患者(肺良性病变组)及34例健康人群(健康组)为本次研究对象.LTA检测采用乳胶凝集法,血清CYFRA21-1、CEA、NSE抗原的检测分别采用放射免疫分析法,测定各TM的阳性率,计算其特异性和诊断率的准确度.结果 肺癌组4项TM阳性率显著高于肺良性病变组和健康组,且P＜0.05.NSCLC患者的LTA阳性率最高、腺癌患者的CEA阳性率最高、鳞癌患者的CYFRA21-1阳性率最高、SCLC患者的NSE阳性率最高,与其他类型患者相比,差异均具有统计学意义P＜0.05.Ⅰ、Ⅱ期肺癌患者4项血清TM阳性率均明显低于Ⅲ期和Ⅳ期,且P＜0.01;4项TM联合检测肺癌阳性率最高为94.31%,特异性也提高到95.22%,准确度也是最高,且4项联合检测可将肺癌早期诊断率阳性率提高到63.37%.结论 不同血清肿瘤标记物、相关抗原对不同类型肺癌的诊断均有价值;LTA、CYFRA21-1、CEA、NSE的联合检测,能有效提高肺癌的检出率,能对早期肺癌的诊断提供辅助依据.     

Early Diagnosis and Lung Cancer Screening

Lung cancer remains the most significant cause of cancer death, accounting for about 20% of all cancer-related mortality. A significant reason for this is delayed diagnosis, either due to lack of symptoms in early-stage disease or presentation with non-specific symptoms common with a broad range of alternative diagnoses. More is needed in terms of increasing public awareness, providing adequate healthcare professional education and implementing clinical pathways that improve the earlier diagnosis of symptomatic lung cancer. Low-dose computed tomography screening of high-risk, asymptomatic populations has been shown to reduce lung cancer mortality, with focus now shifting towards how best to implement lung cancer screening on a wider scale in a safe, efficient and cost-effective manner. For maximum benefit, efforts must be made to optimise uptake, especially among high-risk populations with significant socioeconomic deprivation, as well as successfully incorporate tobacco-dependency treatment. Quality assured programme management will be critical to minimising screening-related harms and adequately managing incidental findings. By undertaking the above, there can be optimism that lung cancer outcomes can be improved significantly in the near future.     

生物标志物在肺癌早筛早诊应用中的研究现状

肺癌是全球范围内恶性程度最高的肿瘤之一.在我国,肺癌的死亡率已连续多年处于首位.早筛早诊是延长肺癌患者生存期的前提.近年来,被认为具有发展前景的液体活检技术逐渐受到关注,其在肺癌早期诊断中的价值值得我们探讨.本文通过综述生物标志物在肺癌筛查和早期诊断中的应用,从多组学寻找特异生物标志物,探讨其在肺癌早期诊断的意义.     

采用抗体库技术筛选抑制肺癌的功能性抗体

 目的 研制抑制肺癌细胞功能性单抗,为治疗肺癌提供靶向治疗剂,并为分离获得肺癌相关的分子靶标打下基础。方法 从新鲜人肺癌组织分离肿瘤细胞免疫Balb/c小鼠,免疫小鼠脾细胞与SP2/0细胞融合后接种于甲基纤维素,制备大容量功能性抗体库。采用活细胞荧光、ELISA、免疫组化、肿瘤增殖、侵袭、黏附、动物体内治疗实验等方法筛选鉴定抑制肿瘤细胞的功能性单抗。结果 本次融合共获得了1573株杂交瘤克隆,在能与肺癌细胞膜反应的314株克隆中,154株与正常肺组织不反应或低反应。功能性筛选发现41株单抗显著地抑制肺癌细胞增殖,24株能抑制肺癌细胞对Matrigel的侵袭,15株能抑制肺癌细胞与CollagenI的黏附,进一步实验验证了2株单抗能在体内抑制肺癌移植瘤的生长。结论 采用大容量功能性抗体库技术成功获得了多株具有抑制肺癌细胞恶性生物学行为的功能性单抗,其中2株可能具有靶向治疗肺癌的应用潜力。     

肺癌生物标记物筛选及在早期诊断中的意义

目的:探讨肺癌生物标记物在肺癌早期诊断中的作用,寻找最佳标记物,提高肺癌诊断水平.方法:随机选取90例肺癌患者及86例健康人,分别检测外周血29种肿瘤标记物.根据检测结果分别计算每一种标记物的特异性、阳性预期值、敏感性、阴性预期值、总有效率及5项均值.结果:29种标记物中按其敏感性和平均值大小得出排序,选取敏感性和5项均值较高的一组为最佳标记群,从5项均值较高的指标PU、SA、CEA、CU-P、CA242、CYFRA等中选取CEA、SA、CA242,敏感性达89.29%.结论:PU、SA、CEA、CA242等可作为肺癌早期诊断最佳标记物组合.     

Breathprinting and Early Diagnosis of Lung Cancer

The electronic nose (e-nose) is a promising technology as a useful addition to the currently available modalities to achieve lung cancer diagnosis. The e-nose can assess the volatile organic compounds detected in the breath and derived from the cellular metabolism. Volatile organic compounds can be analyzed to identify the individual chemical elements as well as their pattern of expression to reproduce a sensorial combination similar to a fingerprint (breathprint). The e-nose can be used alone, mimicking the combinatorial selectivity of the human olfactory system, or as part of a multisensorial platform. This review analyzes the progress made by investigators interested in this technology as well as the perspectives for its future utilization.     

肺癌的分子分期相关研究进展

癌症的分子分期（molecular staging）是指应用各种先进的分子生物学诊断技术检查癌症患者的淋巴结、循环血液及骨髓等组织，从中发现常规影像学或病理组织学无法检测到的隐性微小转移灶，进而从基因或蛋白质水平诊断癌症的转移，根据微转移发生的部位，结合癌症的国际分期标准，最终达到更准确的TNM分期。本文介绍了癌症分子分期的概念．对国内外学者在非小细胞肺癌的分子分期的研究工作进行综述．并提出目前研究中运用的分子生物技术的优势与不足。