论文标题:外周血CK19mRNA表达及CYFRA21-1、CEA、NSE联检对肺癌诊断的研究
The Evaluation of Cytokeratin 19 mRNA Expression And Combination of CYFRA21-1、CEA、NSE in The Peripheral Blood
论文作者 夏怡
论文导师 应可净,论文学位 硕士,论文专业 内科学(呼吸系病)
论文单位 浙江大学,点击次数 258,论文页数 39页File Size1137k
2001-05-01论文网 http://www.lw23.com/lunwen_1243187/ 角蛋白,癌胚抗原,神经元特异性烯醇化酶, 逆转录多聚酶链反应法,循环癌细胞
cytokeratin; carcinoembryonic antigen; neuron-specific enolase; reverse-transcriptase polymerase chain reaction; circulating cancer cell
近半个世纪来，肺癌发病率和死亡率呈持续上升趋势，肺癌死亡率的高低对整个恶性肿瘤的预后举足轻重，因此加强对肺癌诊断的研究尤为重要。目前临床上多以细胞角蛋白21-1片段（CYFRA21-1）、癌胚抗原（CEA）、神经元特异性烯醇化酶（NSE）等肿瘤标志物作为肺癌诊断的辅助检查手段，其对肺癌的灵敏度一般在30-70％之间，此三项肿瘤标志物单项检测均有一定的局限性，联检是否能提高灵敏度报道较少。 肺癌诊断直接影响着肺癌治疗措施的制定，其中远处转移是个关键因素，这使得检测肺癌血源性微转移成为一研究热点。近来随着分子生物学技术的发展，逆转录多聚酶链反应法（RT-PCR）通过快速筛检循环癌细胞来判断是否存在血源性微转移，目前多以细胞角蛋白19（CK19）作为检测上皮性实体癌血源性微转移的靶基因，CK19 mRNA在肺癌组织中有表达已有定论，但其在血细胞中有无表达尚有争论。 为探讨CK19 mRNA是否能作为检测肺癌血源性微转移的靶基因，本研究检测了67例肺癌和30例肺良性疾病（BLD）患者外周血中CK19 mRNA的表达，结果提示肺癌组外周血 CK19 mRNA阳性率显著高于 BLD组。血清CYFRAZI－l＋CEA或CYFRAZI－l＋NSE h项联检能提高对肺癌的灵敏度。 l 病人和方法1＊ 病人：1．1刁病例组为肺癌初诊病人67例，所有病例均经手术或穿刺病理确诊，鳞癌25例，腺癌28例，小细胞肺癌（SCLC）14例。分期根据国际抗癌联盟1997年TNM分期，I期8例，11期15例，Ill期27例，IV期17例。1．1．2对照组为BLD病人30例，肺炎16例，慢性支气管炎4例，肺脓疡1例经临床治疗均排除恶性病变，肺结核2例及肺良性腺瘤7例均经病理确诊。l．2标本采集和 RNA的抽提：第一管静脉血 Zml用于血清 CYFRAZll、CEA、NSE检测。第二管血sml以肝素抗凝，分离出外周血单个有核细胞（PBMCS），Trizol试剂盒抽提总 mRNA，并溶解于 10pl DEPC无菌水。1．3 RT－PCR1．3j RT W系共 20ul，包括 RNA，sx M－MLV RT buffer，0·IM DTT，10mMdNTP，Rnasin，弓物 oligo dT，M－MLV RT，DEPC无菌水，37oC水浴 1J时。设立DEPC无菌水阴性对照组。1．3．2 PCR CK19及卜actin PCR体系共 25PI，包括 cDNA，10x PCR buffer，25mMMgCI。，10mM dNTP，Taq DNA聚合酶，上游引物，下游引物，DEPC无菌水。扩增条件为95 C变性5分钟，再经过40个循环即95C变性1分钟，54oC退火 1分钟，72℃延伸 1，5分钟，最后在 72oC延伸 10分钟，置于 4℃。同时设 2了DEPC无菌水阴性对照组。1．3．3取PCR产物10pl琼脂糖凝胶电泳，紫外荧光数字成像仪观察，CK19mRNA表达阳性者在460hp处有条带出现，在370hp处出现的条带为6咆ctin产物。1．4血清 CYFRAZI－l，CEA，NSE测定 一 血清CYFRAZI－1用酶联免疫吸附法测定，＞3．3ng／thl为阳性。CEA用时间分辨荧光法测定，＞sng／ml为阳性。NSE用酶联兔疫吸附法测定，＞13ng／ml为阳性。1．S统计方法：用SSPS10．0统计软件包进行分析处理。2结果2、l肺癌组 CK19 mRNA阳性率为 57．6％，对照组为 33，3O，两组比较有显著差异（P＜0．05）。对照组中，男与女之间或吸烟者与不吸烟者之间 CK19 mRNA阳性率无显著差异，CK19 mRNA阳性组与阴性组病人平均年龄无显著差异。2．2肺癌组中鳞癌 CK19 mRNA阳性率为 60．0％，腺癌为 50．0％，非小细胞肺癌（NSqLC）为 54厂％，SCLC为 64．3％，各组织类型间 CK19 mRNA阳性率比较无显著差异（P＞0．05）。2．3肺癌 1＋11期 CK19 mRNA阳性率为 30．4O，Ill、W期分别为 70．4％及 70．6％，Ill、IV期CK19 mANA阳性率显著高于1八期（P＜0．of）。2，4肺癌组与BLD组CYFRAZI－l、CEA、NSE浓度水平比较有显著差异。2．5 NSCLC血清 CYFRAZI－l、CEA水平及阳性率显著高于 SCLC，鳞癌CYFRAZI－l、腺癌 CEA、SCLC NSE水平及阳性率显著高于其他类型。26 Ill、N期肺癌 CYFRAZI－l。CEA、NSE水平及阳性率显著高于 1＋11期。 327 CYFRA21刁＋CEA或CYFRAZll＋NSE二项联检对肺癌的灵敏度从单项的28．4－52．20提升到70．lO，显著高于单项检测（P＜0．05），特异性无明显下降 （Ptoo．05）。三项联检的灵敏度从二项联检的70＊％提升到79．l％，并不显著高于二项联检（P＞0．05）。四项联检灵敏度显著高于二项联检，特异性亦明显下降（P＜0．05）。NSCLC中，CYFRA21－l＋CEA联检的灵敏度从单项的 20．7 一 60．4O升至 79．2％，显著高于单项检测（P＜0．05），特异性无明显下降（PTh0．05）；sCLC中，二项及三项联检灵敏度并不显著高于NsE单项检测（P＞0刀5）。3．结论3刁 以 CK mRNA为靶基因的 RT．PCR法能检测出肺癌外周血中 CKmRNA的表达，可作为检测肺癌血源性微转移的靶基因，但特异性不够高?
Lung cancer is one of the most frequent malignant tumors in humans and the prognosis is poor. In order to improve the clinical approach to lung cancer patients, several serum tumor markers such as CYFRA2 1-1 CEA NSE have been studied. The sensitivity of each of these markers is not high enough, the study about combination ofCYFRA2I-1. CEA and NSE is few. Distant metastases are a key factor in planning the treament of lung cancer, so recent interest has been focused on the detection of circulating cancer cells in the peripheral blood of lung cancer patients. Now a technique named reverse -transcriptase polymerase chain reaction (RT-PCR) has been used to detect the hematogenous micrometastases. The sensitivity and specificity of this techinique are higher than immunoctology. Cytokeratin 19 (CKI9) is used as a target gene. Immunologic studies have been showed CK19 mRNA expression in a significant 5 number of lung cancer specimens, but low-level expression of CKI9 mRNA in mormal peripheral blood mononucleated cells (PBMCS) is still debated. Based on these data, we analyzed expression of CK19 mRNA in PBMCS from 67 lung cancer patients and 30 benign lung diseases (BLD) .The rate of CK 19 mRNA positivity was significant higher in lung cancer patients than in BLD controls (P < 0.05). Combination of CYFRA2I-1 and CEA or NSE can improve the sensitivity. 1 Patients and Methods 1.1 Patients 1.1.1 The study population consisted of 67 histologically documoned lung cancers. Staging procedures were based on TNM stage in 1997 of UICC, 23 stage I + II, 27 stage III, 17 stage IV. Histopathologic examination showed 25 squamous cell cancinomas, 28 adenocarcinomas, 14 small-celIl lung cancers (SCLC). 1.1.2 The control population consisted of 30 BLD, it contained pneumonias, chronic bronchitis, lung abscess, pulmonary tuberculosis, lung benign tumor. 1.2 Sample Collection and RNA Preparation We used Vacutainer collection tubes to collect 7ml blood, 2m1 blood in the first tube was used to detect CYFRA21-1 CEA and NSE, 5m1 blood in the second tube containing heparin was used to detect CKI 9 mRNA. then used Trizol Kit to extract total mRNA from PBMCS and solved it in I Opi DEPC water. 1.3 RT-PCR 1.3.1 RT 6 2OiiI reaction mix contained oligo dT primer, 10mM deoxynucleotide trisphophate (dNTP) , 0.1 M dithiothreitol (DTT) , RNAs in, MMLV-RT, 5 x MMLV-RT buffer, DEPC water. Negative control contained DEPC water instead of RNA. 1.3.2 PCR PCR reactions were performed in 25.tl final volume that contained cDNA, lOx PCR buffer, 25mM Mgcl2, 10mM dNTP, Taq polymerase, primers for CK19 and ~ -actin . PCR conditions were consisted of one amplification round of 40 cycles at 950C for 1 minute, 54擟 for 1 minute, 72扖 for 90 seconds and 720C for 1 Ominutes . Negative control contained DEPC water instead of cDNA. 1.3.3 After the PCR was completed, 1 Oi.tl of PCR product were analyzed on agarose gel . CK19 produt was 460 bp size and ~ -actin produt was 370 bp size. 1.4 CYFRA2 1-1 and NS
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