【作者】 郑磊；

【导师】 吴育连；

【作者基本信息】 浙江大学 ， 外科学（普外科）（专业学位）， 2020， 博士

【摘要】 背景:胰腺癌(Pancreatic cancer,PC)在消化系统中属于恶性程度很高的肿瘤。早期胰腺癌由于其非典型的临床症状以及早期筛查指标缺乏特异性,通常难以诊断,从而失去了进行根治性手术切除的机会。而晚期胰腺癌对临床放射疗法和化学疗法的敏感性较低,而且放化疗副作用对病人身体影响较大,胰腺癌患者的预后很差。我们想通过提高抗肿瘤药物的靶向性,从而提高抗肿瘤药物对胰腺癌的疗效并降低对机体正常细胞的损伤。在靶向性问题上,我们利用噬菌体展示技术进行筛选。在抗肿瘤药物上,我们选择了KLA((KLAKLAK)2)凋亡肽作为治疗肽段,KLA凋亡肽段与化学治疗药物相比,具有分子量小、肿瘤穿透能力强、免疫原性低、生物相容性好、毒性作用小、易于合成和修饰等优点[12-13],是理想的抗肿瘤药物。但KLA凋亡肽很难透过细胞膜,无法进入细胞后就不能破坏线粒体膜以诱导细胞凋亡[16]。因此本研究旨在通过利用噬菌体展示技术,筛选出能特异性作用于胰腺癌细胞的靶向肽段。利用该肽段的靶向性,实现对胰腺癌的影像学诊断。并利用该肽段结合凋亡肽段KLA,以期实现融合肽段对胰腺癌细胞具有靶向性并对胰腺癌细胞及肿瘤的生长有抑制作用,并对其相关作用机制进行探讨。方法:本研究选取人源胰腺癌细胞系PANC-1作为靶细胞。首先,我们利用噬菌体展示技术筛选出靶向PANC-1细胞的噬菌体。接着,将筛选到的噬菌体进行扩增后、DNA提取,然后测序。通过与试剂盒提供的简要遗传密码表进行比对,推算出氨基酸序列。利用得到的氨基酸序列合成靶向肽段HMNPWSD,用异硫氰酸荧光素(fluoresceinisothiocyanate,FITC)标记,并与PANC-1细胞共孵育后,利用细胞免疫荧光实验及流式细胞仪对其靶向性进行了评估。然后,将靶向肽段HMNPWSD与KLA肽段进行了共轭,并再次利用细胞免疫荧光实验及小动物活体成像系统(In vivo imaging system,IVIS)对融合肽段HMNPWSD-KLA进行了体外细胞及体内肿瘤的靶向性评估实验。我们通过MTT法检测了HMNPWSD-KLA融合肽段对PANC-1细胞增殖功能的影响;并利用流式细胞凋亡分析、Western Blot以及线粒体膜电位评估等方法对其抑制PANC-1细胞生长机制进行了研究。最后我们建立了荷载PANC-1肿瘤的裸鼠模型,然后,通过动物实验观察了融合肽段HMNPWSD-KLA在体内对PANC-1肿瘤生长影响。并通过裸鼠器官组织切片及相关血液指标对融合肽段HMNPWSD-KLA的副作用进行了评估。结果:我们通过噬菌体展示技术筛选到的靶向PANC-1细胞的肽段HMNPWSD,细胞免疫荧光实验及流式细胞荧光分析实验结果验证了其对PANC-1细胞具有特异性。细胞免疫荧光及IVIS实验结果证实了融合肽段HMNPWSD-KLA对PANC-1细胞及肿瘤的靶向性。MTT法证实了融合肽段对PANC-1细胞的生长增殖抑制作用。流式细胞凋亡分析、Western Blot以及线粒体膜电位评估等结果证实了对其抑制PANC-1细胞生长机制是通过破坏细胞线粒体,诱导细胞凋亡。利用融合肽段HMNPWSD-KLA作用于荷载PANC-1肿瘤的裸鼠模型实验结果证明了融合肽段对PANC-1肿瘤生长起到明显抑制作用。此外,通过检测裸鼠的器官组织切片及血液学参数证明融合肽段对裸鼠机体影响不大。结论:融合肽段HMNPWSD-KLA对胰腺癌细胞系PANC-1细胞及肿瘤具有靶向特异性,而且该融合肽段通过破坏PANC-1细胞线粒体,诱导细胞凋亡,对细胞及肿瘤的生长起到了明显抑制作用。此外,该融合肽段对于裸鼠重要器官及血液学参数影响不大。更多还原

【Abstract】 Background: Pancreatic cancer(PC)is a highly malignant tumor of the digestive system.Early pancreatic cancer is often difficult to diagnose due to its atypical clinical symptoms and lack of specificity for early screening indicators,thus losing the opportunity for radical surgical resection.Advanced pancreatic cancer is less sensitive to clinical radiation therapy and chemotherapy,and the side effects of radiotherapy and chemotherapy have a greater impact on the patient’s body.The prognosis of patients with pancreatic cancer is poor.We want to increase the targeting of anti-tumor drugs to improve the efficacy of anti-tumor drugs on pancreatic cancer and reduce damage to normal cells in the body.On the issue of targeting,we use phage display technology for screening.For anti-tumor drugs,we selected KLA((KLAKLAK)2)apoptotic peptide as a therapeutic peptide.Compared with chemotherapeutic drugs,KLA apoptotic peptide has a small molecular weight,strong tumor penetration ability,and low immunogenicity,good biocompatibility,low toxicity,easy synthesis and modification[12-13],is an ideal anti-tumor drug.However,KLA apoptotic peptides are difficult to penetrate the cell membrane,and after entering the cell,they cannot destroy the mitochondrial membrane to induce apoptosis [16].Therefore,the purpose of this study is to use phage display technology to screen out targeted peptides that can specifically act on pancreatic cancer cells.The targeting of the peptide is used to realize imaging diagnosis of pancreatic cancer.And the peptide is combined with the apoptotic peptide KLA,in order to realize that the fusion peptide is targeted to pancreatic cancer cells and inhibits the growth of pancreatic cancer cells and tumors,and the related mechanism was studied.Methods: In this study,human pancreatic cancer cell line PANC-1 was selected as the target cell.First,we used phage display technology to screen out phages that targeted PANC-1 cells.Second,the screened phages were amplified,DNA was extracted and sequenced.The amino acid sequence was deduced by comparison with the brief genetic code table provided by the kit.The targeting peptide HMNPWSD was synthesized according to the obtained amino acid sequence.The peptide was labeled with fluoresceinisothiocyanate(FITC)and incubated with PANC-1 cells.Its affinity was evaluated by using cell immunofluorescence experiments and flow cytometry.The target peptide HMNPWSD was conjugated with the KLA peptide,and the cell immunofluorescence experiment and in vivo imaging system(IVIS)were used to assess the affinity of target peptide for the cell in vitro and Tumors in vivo.The effect of HMNPWSD-KLA fusion peptide on the proliferation function of PANC-1 cells was test by MTT assay.Flow cytometry analysis,Western Blot,and mitochondrial membrane potential assessment were performed to study the mechanism of PANC-1 cell growth inhibition.Finally,we established a nude mouse model bearing PANC-1 tumors,and then observed the effects of the fusion peptide HMNPWSD-KLA on the growth of PANC-1 tumors in vivo through animal experiments.The side effects of the fusion peptide HMNPWSD-KLA were evaluated by organ tissue sections and related hematological parameters of nude mice.Results: We screened out the peptide HMNPWSD targeting PANC-1 cells by phage display technology,and the results of cell immunofluorescence and flow cytometry analysis confirmed its specificity for PANC-1 cells.At the same time,the specific affinity of the fusion peptide HMNPWSD-KLA to PANC-1 cells and tumors was confirmed by immunofluorescence and IVIS experiments.The inhibitory effect of the fusion peptide on the growth and proliferation of PANC-1 cells was verified by the MTT assay.The result of flow cytometry analysis,Western Blot,and mitochondrial membrane potential evaluation prove that the mechanism for inhibiting the growth of PANC-1 cells is through the destruction of cell mitochondria and inducing apoptosis.The experimental results of a nude mouse model bearing the PANC-1 tumor proved that the fusion peptide can significantly inhibit the growth of PANC-1 tumor.In addition,it has been proved that the fusion peptide has little effect on nude mice by examining organ tissue sections and hematological parameters of nude mice.Conclusion: The fusion peptide HMNPWSD-KLA has targeting specificity for pancreatic cancer cell line PANC-1 cells and tumors.Moreover,the fusion peptide induces apoptosis by destroying the mitochondria of PANC-1 cells,which significantly inhibits the growth of cells and tumors.In addition,the fusion peptide has little side effects on important organs and hematological parameters of nude mice.更多还原