摘要
肺癌是全球最普遍的恶性肿瘤之一,其发病率和病死率均占各种恶性肿瘤之首。在我国,近20多年来肺癌发病和死亡呈迅速上升趋势,中国癌症预防与控制规划纲要(2004-2010)表明肺癌已成为我国的第一大癌症。目前,肺癌从确诊到死亡的五年存活率仍不到15%,其主要原因之一是临床确诊的肺癌病人往往已经处于病程中晚期或发生了癌细胞的远端转移。因此,实现早期诊断、早期治疗是治疗肺癌的首选策略。噬菌体展示肽库技术作为探索蛋白之间相互作用的工具将大量随机多肽与其DNA编码序列之间建立了直接的联系,使得各种靶分子的多肽配体通过淘选的程序得以快速鉴定,已成为研究蛋白质分子之间相互作用的有效工具。它具有简便、高效、大通量筛选的优点,并可以通过宿主菌扩增与低表达量的特异性分子结合的噬菌体克隆,已广泛应用于抗肿瘤药物、肿瘤诊断标志物等的筛选和研发。
鉴于此,我们利用噬菌体展示十二肽库与肺鳞癌细胞NCI-H1299和肺正常细胞MRC-5进行3轮全细胞减性筛选,3轮筛选后与NCI-H1299细胞特异性结合的噬菌体富集了100倍。我们从第3轮筛得的噬菌体中随机挑取120个单克隆,同时从原库中随机挑取噬菌体单克隆做为对照,采用ELISA及细胞免疫化学从噬菌体水平初步鉴定噬菌体单克隆对NCI-H1299的亲和力。ELISA结果显示,45个噬菌体单克隆对NCI-H1299有较的高亲和力(P/N>2),阳性率为37.5%;其中,P/N>2.2的有24个,细胞免疫化学结果显示它们与肺癌细胞的结合呈显著阳性,结合部位主要在细胞膜上。
我们将P/N>2.2的24个噬菌体单克隆扩增,提取质粒后测序。测序结果显示,24个噬菌体单克隆单链DNA中插入的外源序列为10条不同的核苷酸序列,我们将这10条序列分别命名为zs1、zs2……zs10。其中zs6出现了13次,重复的频率最高,达到了54.2%;zs6编码由3个Thr (T),2个Leu (L),2个His (H),1个Ala (A),1个Asp (D),1个Arg (R),1个Pro (P),1个Trp (W)组成的12肽,其氨基酸序列为**AD***HPPW*。该序列与包括肺癌在内的恶性肿瘤的关系尚未见文献报道。
我们通过化学方法合成多肽zs6并部分标记绿色荧光素异硫氰酸(FITC)。首先,利用细胞免疫荧光、组织免疫荧光鉴定多肽[zs6]FITC与肺癌细胞、人肺癌组织结合的特异性和亲和力。细胞免疫荧光实验结果显示[zs6]FITC对肺鳞癌细胞NCI-H1299具有高亲和力,并且对肺腺癌细胞A549同样具有较高亲和力,但与肺正常细胞MRC-5结合能力很弱。组织免疫荧光实验结果显示[zs6]FITC与人肺癌组织的结合能力显著高于肺正常组织的;同时,人肺癌组织芯片实验结果显示,[zs6]FITC用于临床肺癌鉴定的阳性率可达到24.8%。然后,通过多肽[zs6]FITC在荷瘤裸鼠体内的分布实验观察[zs6]FITC在荷人肺癌A549裸鼠体内的分布情况及其稳定性。实验结果显示[zs6]FITC在肿瘤部位分布较多,有较好的靶向性,其次,在主要代谢器官肝脏、肾脏组织也有分布,但在裸鼠大脑、心脏、肺脏等正常组织则未见及分布。
为了进一步研究多肽zs6与肺癌的结合位点,我们通过化学方法合成生物素(Biotin)偶联的多肽zs6,将[zs6] Biotin固定在亲和素(avidin)包被的酶标板板上,与人肺癌cDNA文库进行4轮筛选,挑取单克隆PCR扩增、纯化后进行测序。对序列进行生物信息学分析,发现与该多肽特异性结合的肺癌抗原有Envoplakin、ATPase和987bp的未知蛋白。(1)目前,未见Envoplakin与肺癌相关的任何报道。生物信息学分析发现Envoplakin在非小细胞肺癌(腺癌和鳞癌)有大量表达,而在小细胞肺癌则表达量较少。因此,可能可以作为非小细胞肺癌(腺癌和鳞癌)的诊断标志物。(2)ATPase广泛存在与已知的各种生命形式,对肺癌研究不具有特异性价值;(3)987bp的未知序列经生物信息学分析发现其与人类基因及基因组未见同源性,表明我们可能发现了新的肿瘤特有的抗原,确认其在肺癌与正常组织中的差异将阐明一新的肺癌诊断和治疗靶点,为肺癌的早期诊断和靶向性治疗奠定基础。
Lung cancer is one of the most common malignant tumor in the worldwide, and it’s morbidity and mortality are both on the first rank of all malignant tumors. In China, it’s morbidity and mortality showed a rapid increase during the past 20 years. Chinese Cancer Prevention and Control Program (2004-2010) showed that lung cancer has become the most common cancer in China. So far, the five-year survival rate of lung cancer is less than 15% from diagnosis to death. One of the major reason is that the lung cancer patients who have been definited are often already in advanced stage or metastasis. Therefore, early diagnosis and early treatment are the preferred strategy for the treatment of lung cancer. Phage display peptide library as a powerful tool can be used to explore the interaction during proteins. It establish direct connections between large numbers of random peptides and DNA. Beacause of convenient and effective, it has been widely used in screening and researching anticancer drugs and tumor biomarkers.
The lung squamous carcinoma cell line NCI-H1299 was used as the antigen and lung normal cell line MRC-5 was used as control for subtraction biopanning from Ph.D.-12TM phage display peptide library. After 3 rounds of panning, 106 phage clones have been received. The enrichment of phages which are binding with NCI-H1299 is about 100-fold. Then, 120 phage clones were randomly selected from the 106 phage clones and amplified. Meanwhile, some phage clones were picked up from the original library as the control. The affinity and specificity with NCI-H1299 of phage clones were identified by ELISA and immunocytochemical staining. ELISA results showed that 45 phage clones had a high affinity to NCI-H1299(P/N>2), the positive rate was 37.5%. Besides, 24 of 45 showed more affinity to NCI-H1299 (P/N>2.2) and DAB staining results confirmed they could specifically bind on the surface of NCI-H1299.
The 24 phage positive clones were amplified and sequenced. 10 different nucleotide sequence were obtained and named zs1, zs2…zs10 separately. Among these sequence, zs6 repeated 13 times. It was the only sequence which repeated most frequently and the repeat rate was 54.2%. It encod a 12 peptide consisted by the 3 Thr (T), 2 Leu (L), 2 His (H), 1 Ala (A), 1 Asp (D), 1 Arg (R), 1 Pro (P), 1 Trp (W), the amino acid sequence is **AD***HRPW*. zs6 has not yet been reported in any literature. There is no envidence showed there is any relationships between zs6 and tumors including lung cancer.
Subsequently, the peptide zs6 and fluorescein isothiocyanate(FITC)-conjugated peptide zs6 were synthesized and purified. First, immunofluorescence assay was used to identify the specificity and affinity of peptide [zs6]FITC to lung cancer cells and human lung cancer tissue. The results showed that peptide [zs6]FITC has high affinity with lung squamous carcinoma cell line NCI-H1299 and lung adenocarcinoma cell line A549, but almost no affinity with normal lung cell line MRC-5. Meanwhile, the affiniy of peptide [zs6]FITC with human lung cancer tissue was significantly higher than normal tissues. At the same time, human lung cancer tissue microarray experimental results show that the positive rate of peptide [zs6]FITC using for identification of human lung cancer tissue was 24.8%. Then, tumor-bearing nude mice experiment was used to observe the distribution of peptide [zs6]FITC in vivo. It suggest that peptide zs6 could bind specifically to lung tumor in vivo and there were no distribution in nude mice brain, heart, lung and other normal tissues. But the peptide [zs6]FITC still can be found in the major metabolic organ, such as liver, kidney.
In order to search the binding site of peptide zs6 with lung cancer, Biotin- conjugated peptide zs6 was synthesized and purified. Peptide [zs6]Biotin was immobilized on ELISA plate which has coated with avidin. Then, peptide zs6 was panning with human lung cDNA library. After 4 rounds screening, several phage clones were picked and amplified. Plasmid of phage were extracted and amplified by PCR, then the PCR products were purified and sequenced. Bioinformatics analysis results revealed that the peptide-specific lung cancer antigens are Envoplakin, ATPase and 987bp unknown proteins. (1)At present, there is no reports about the relationships between Envoplakin and lung cancer. Bioinformatic analysis found that Envoplakin have a higher expression in non-small cell lung cancer (adenocarcinoma and squamous cell carcinoma) and a lower expression in small-cell lung cancer. Thus, Envoplakin may be used as a diagnostic markers of non-small cell lung cancer (adenocarcinoma and squamous cell carcinoma). (2) ATPase exist in a variety of life forms. So the study of it do not have specific values; (3) Bioinformatics analysis showed that 987bp sequence has no homologous with the human genome. It indicated that we may have found a new tumor-specific antigen. If the expression of it have difference between lung cancer and normal tissues, it may used as a new target for diagnosis and treatment of lung cancer and lay a foundation for targeting therapy.
引文
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