【作者】 潘琴；

【导师】 冯冰虹； 臧林泉；

【作者基本信息】 广东药学院 ， 药剂学， 2009， 硕士

【摘要】 肺癌是世界范围内最常见、致死人数最多的恶性肿瘤之一[1],可分为小细胞肺癌(small cell lung cancer,SCLC)和非小细胞肺癌(non-small cell lung cancer,NSCLC)两种,其中非小细胞肺癌约占肺癌总数的80%。由于肺癌起病隐匿,肺癌的筛查和治疗方面没有得到根本的改善,患者出现症状时多为晚期,预后较差,肺癌的5年存活率仅为13%;80%的患者在诊断后1年内死亡。而肺癌的早期诊断患者的5年存活率可达60%～90%[2]。因此,积极寻找肺癌早期诊断的特异性标志物是提高肺癌治疗效果的根本所在;同时,对于中晚期肺癌,加速靶向治疗药物的研发是克服当前肺癌化疗高毒、低疗效的有效手段。目的:以肺癌细胞A549为研究目标,利用体内噬菌体展示技术筛选出与肺癌特异性结合的多肽,为肺癌的早期诊断和靶向治疗奠定基础。方法:首先,我们将肺癌细胞A549接种于裸鼠体内,待其荷瘤成功并增殖到一定程度,采用体内噬菌体肽库展示技术,将肽库静脉注射入荷瘤裸鼠体内,然后筛选与肺癌组织特异性结合的含外源多肽的噬菌体,经过3轮体内筛选。然后,随机挑取一批第3轮筛选后的噬菌体单克隆,采用ELISA及细胞免疫化学鉴定噬菌体对肺癌细胞A549的亲和力。挑取阳性克隆扩增,按照分子克隆上的方法提取噬菌体单链DNA,送测序公司进行序列测定,根据测序结果获得各噬菌体外源多肽的核苷酸序列,对获得的多肽进行生物信息学分析。接下来进行多肽的特异性鉴定,首先,通过化学合成多肽并标记荧光FITC。然后,利用荧光标记多肽鉴定多肽与肺癌细胞及组织结合的特异性、亲和力、体内分布等。利用细胞免疫荧光鉴定多肽对肺癌细胞A549、NCI-H1299、肝癌细胞7402、HepG2、肺正常细胞WI-38、肝正常细胞LO2的亲和力。利用组织免疫荧光鉴定多肽对肺癌组织、肺正常组织的结合。利用荧光多肽在荷瘤裸鼠体内分布实验鉴定多肽在荷瘤裸鼠的靶向性分布。结果:经3轮体内筛选,噬菌体在肿瘤组织中富集了104倍。随机挑取第3轮筛选后的30个噬菌体单克隆,分别命名为phage-1、phage-2、…,phage-30,采用ELISA鉴定噬菌体对肺癌细胞A549的亲和力,成功获得了对A549有较高亲和力噬菌体克隆27个,其中,P/N > 2.5以上的有17个克隆,而在上述17个克隆中,P/N > 3.0的克隆有4个,分别是phage-1、phage-12、phage-14和phage-20,同时,细胞免疫化学鉴定这4个克隆对A549的亲和力,与正常对照组相比,DAB染色呈显著阳性。将ELISA鉴定后的P/N > 2.5的噬菌体克隆共17个扩增,测序,根据测序结果获得噬菌体外源多肽的核苷酸序列,其中有14个噬菌体克隆分别插入了6条不同的外源多肽核苷酸序列,分别命名为zp1, zp2, zp3, zp4, zp5, zp6,另外还有3个噬菌体克隆测序结果显示其外源多肽序列丢失(空噬菌体)。测序结果显示多肽zp2重复频率最高,达到50%,且生物信息学分析其在国内外文献尚未见报道,其功能也属未知,故我们拟对多肽zp2进行系统研究。通过化学合成多肽zp2全长,并标记荧光FITC(zp2-FITC)。细胞免疫荧光实验显示,zp2-FITC对肺腺癌细胞A549具有较强亲和力,并且也发现,其对肺鳞癌细胞NCI-H1299同样有较强亲和力,对肝癌细胞7402、HepG2的结合较弱,但对肺正常细胞WI-38、肝正常细胞LO2亲和力不强。组织免疫荧光实验显示,zp2-FITC对临床肺癌组织的结合显著高于正常对照组。裸鼠体内分布实验显示,zp2-FITC在荷瘤裸鼠肿瘤部位分布较多,有较好的靶向性,其次,在肝脏、肾脏组织也有分布,可能与其是主要的代谢器官有关,但是在裸鼠大脑、心脏、肺脏等正常组织未见及分布。结论:(1)成功建立了裸鼠荷瘤模型;(2)利用上述模型,在体内成功筛选出与肺癌组织特异性结合的噬菌体;(3)利用ELISA和细胞免疫化学鉴定出27个噬菌体单克隆对A549有高度特异性和亲和力;(4)经过DNA测序,获得了6条多肽序列,分别命名为zp1、zp2…zp6,经生物信息学分析发现,zp2尚未见研究报道,本课题对zp2进行了系统研究;(5)利用细胞免疫荧光和组织免疫荧光鉴定了多肽zp2对人肺癌细胞、组织具有较高特异性和亲和力,裸鼠体内分布实验显示多肽zp2具有显著的肿瘤靶向性。更多还原

【Abstract】 Lung cancer divided into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), non-small cell lung cancer accounts for about 80 percent of the total number of lung cancer. Lung cancer is one of the world’s malignant tumor becase of its highest morbidity and mortality, mainly due to lung cancer onset of occultd and the screening and treatment is no fundamental improvement. when lung cancer symptoms that the cancer is usually advanced, and poor prognosis with a total of five-year survival rate not more than 15 percent and symptomatic persons less than 10 percent. However, in the early diagnosis of lung cancer patients, the prognosis of surgical treatment in a marked improvement than advanced lung cancer, and the survival rate up to 70 percent. Therefore, actively looking for early diagnosis of lung cancer-specific markers is the most important problem to improve of lung cancer treatment. At the same time, for advanced lung cancer, to accelerate the development of targeted therapy drugs are an effective means to overcome the current highly toxic cancer chemotherapy, low-efficacy, easy-resistant.Objective: lung cancer cells A549 as the study target, using phage display technology in vivo panning and identificated peptides which combined specific with the lung cancer, which lay the foundation for the early diagnosis and targeted therapy of lung cancer. Methods: First of all, the lung cancer cells A549 were inoculated into nude mice when the tumors to a certain degree of value-added, then,using phage display technology in vivo, peptide library was injected into tumor-bearing vein in nude mice, and then get lung cancer with combination of tissue-specific exogenous phage peptides, to do so in vivo screening of three rounds.Then, randomly selected a group of monoclonal phage from the third round panning, using ELISA and Cell-mediated immunity chemistry to identificated the affinity to A549 lung cancer cells.Amplification of positive clones picked, then extracted phage single-stranded DNA according to the method of molecular cloning, which send to sequencing companies ,then the phage peptides exogenous nucleotide sequence were obtained according to sequencing results, and then the sequences were Bioinformatic analysised.Next to the specific identification of peptides, first chemical synthesis peptides, and fluorescence labeling FITC. Using fluorescent-labeled peptides to identified the specificity, affinity, in vivo distribution of peptide with lung cancer cells and tissue. Cell Immunofluorescence identification of peptide’s affinity on lung cancer cell A549, NCI-H1299, hepatoma cell 7402, HepG2, normal lung cell WI-38, and normal liver cell LO2. Tissue Immunofluorescence identification of peptide’s affinity on lung cancer tissue , lung normal tissue. The use of fluorescent peptides in the distribution of tumor-bearing nude mice in the experimental identification of peptides targeting distribution in the tumor-bearing nude mice.Results: After three rounds panning in vivo, the phage which combined with the tumor tissue is 104-fold enriched. Then randomly selected 30 monoclonal phages from the third round screening, ELISA were used to identified the affinity of the phages to lung cancer cells A549. ELISA results shows that 27 phage clones have high-affinity to A549 in all the 30 phage clones, in which, P/N> 2.5 more than 17 clones, and 17 in the above-mentioned clones, P/N> 3.0 have four clones , namely, 1, 12, 14 and 20 clones, at the same time, Cell-mediated immunity chemical were used to identify the affinity of the clones which P/N> 3.0 to A549 , compared with normal control group, DAB staining was significantly positive. Subsequently, 17 phage clones which identified by ELISA with the results P/N> 2.5 were amplified and sequenced, then the phage peptides exogenous nucleotide sequence were obtained according to sequencing results, of which 14 phage clones were inserted into the 6 different exogenous peptide nucleotide sequence, named zp1, zp2, zp3, zp4, zp5, zp6, the other 3 phage clones show that the loss of peptide sequence (empty phage).Bioinformatics analysis showed that the polypeptide of the amino acid sequence zp2 literature at home and abroad, has yet to report, and its function is unknown, so we intend to carry out zp2 peptide in system. Next peptide zp2 length was chemical syntheticed, and fluorescent labeling FITC (zp2-FITC). Cell Immuno- -fluorescence experiments showed that zp2-FITC has a strong affinity on A549, and also on NCI-H1299 , and that affinity was weak on 7402 and HepG2 ,However normal lung cells WI-38, normal liver cells LO2 don’t have. Tissue Immuno- -fluorescence experiments showed that zp2-FITC on the combination of clinical lung cancer tissues was significantly higher than the normal control group. Experiments showed that the distribution of nude mice, zp2-FITC in tumor-bearing nude mice more tumor site distribution, better targeting, and secondly, in the liver, kidney organizations have distribution, however, in nude mice brain, heart, lungs, etc. normal tissues and their distribution is not.Conclusion: (1) successfully established tumor-bearing nude mice model; (2)Panning the lung cancer-specific phage in vivo by using the above-mentioned models; (3) Using ELISA and cell-mediated immunity chemical identified 27 phage clones has a high degree of specificity and affinity to A549; (4) After DNA sequencing, obtaining more than 6 peptide sequence, respectively, named zp1, zp2 ... zp6, by bioinformatics analysis, zp2 study has not yet been reported, in the subject zp2 to be take for systematic studies; (5) Using immunocytochemistry and immunohistochemistry identified peptide zp2 has high specificity and affinity to human lung cancer cells and tissues,and certificated peptide zp2 has significant tumor targeting;更多还原