新生血管亲和性多肽的筛选及功能化脂质体的肿瘤靶向研究

发布时间：2018-03-11 18:41

  本文选题：多肽探针　切入点：VEGFR2　出处：《锦州医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的化疗是目前癌症治疗中常用的手段,但传统的化疗药物仍面临着许多亟待解决的问题,如体内生物利用度低、溶解性差、严重的不良副作用以及易产生耐药性等。近年来,将纳米技术应用于癌症的治疗在一定程度上增加了化疗药物的疗效,但是仍然存在一些不足,如药物不可控的释放、肿瘤病灶靶向性低等。然而,功能性靶向药物递送系统可以有效的解决常规纳米药物载体存在的一些问题,如在一个纳米体系中可实现高效的肿瘤靶向、药物控制释放等多种功能。为此,我们构建了一种新颖的靶向新生血管生成的抗肿瘤纳米药物载体(S1-LS),旨在提高小分子化疗药物的抗肿瘤疗效,同时也能够减小药物对其他正常组织造成的毒副作用。方法针对血管内皮细胞生长因子受体2(VEGFR2),通过多肽固相合成技术、利用混合均分法构建“一珠一物”(OBOC)多肽文库。随后,利用免疫磁珠的方法筛选阳性肽珠,同时通过微阵列芯片结合基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)对阳性多肽进行原位检测分析,Clustal X2多重序列对比分析筛选获得最优多肽序列。分别在分子和细胞水平上验证多肽的亲和性和特异性。最后,采用薄膜分散法制备VEGFR2靶向纳米药物载体(S1-LS),并在体外细胞水平以人脐静脉血管内皮细胞(HUVEC)为阳性细胞模型以及动物水平以人结肠癌(HT-29)荷瘤小鼠为模型评价S1-LS靶向递送药物的效率。结果微阵列芯片系统从所构建106多肽文库中捕获400多个阳性肽珠。原位裂解、测序被捕获的肽珠-VEGFR2-生物素-链霉亲和素-磁珠桥连的磁性肽珠后,通过序列对比分析筛选获得VEGFR2高亲和性多肽S1(Leu-Ile-Asn-His-Glu-Trp-LysGlu-Asn-Tyr-Phe-Pro-Leu-Ser-Phe)。S1与VEGFR2在分子水平的亲和性由表面等离子共振成像系统(SPRi)测得(KD=1.31×10-7 M)。在细胞水平上,通过激光共聚焦显微镜定位异硫氰酸荧光素(FITC)标记的S1(S1-FITC),证明了VEGFR2过表达的阳性细胞(HUVEC)与S1具有高的结合效率。S1成功修饰两亲性分子后制备得到大小均一、分散均匀的VEGFR2-靶向纳米脂质体S1-LS。S1-LS在体外阳性细胞(HUVEC)及体内动物肿瘤模型(HT-29荷瘤小鼠)的实验中都显示了较高的药物靶向递送效率。结论利用优化的微阵列芯片成功进行了VEGFR2-靶向多肽的高效、高通量的筛选和鉴定;且在优化多肽分子探针后成功构建了特异性靶向纳米药物递送系统(S1-LS)。基于VEGFR2靶向多肽S1修饰的纳米脂质体(S1-LS)成功实现了高效的肿瘤靶向药物递送。S1-LS有望被用于癌症的靶向诊断和治疗中,为实现精准化诊断和治疗提供新思路、新方法。
[Abstract]:Objective chemotherapy is a commonly used method in the treatment of cancer at present, but the traditional chemotherapeutic drugs still face many problems to be solved urgently, such as low bioavailability in vivo, poor solubility, serious adverse side effects and easy to produce drug resistance. The application of nanotechnology to the treatment of cancer increases the efficacy of chemotherapeutic drugs to some extent, but there are still some shortcomings, such as the uncontrolled release of drugs, the low targeting of tumor lesions, etc. However, Functional targeting drug delivery systems can effectively solve some of the problems existing in conventional nano-drug carriers, such as the realization of high efficiency tumor targeting, drug release control and other functions in a nano-system. We constructed a novel anti-tumor nano-drug carrier, S1-LSN, targeting angiogenesis, in order to improve the antitumor effect of small molecular chemotherapeutic drugs. Methods for vascular endothelial growth factor receptor 2VEGFR2, the peptide library of "one bead, one object" was constructed by using the technique of peptide solid phase synthesis. Positive peptide beads were screened by immunomagnetic beads. At the same time, in situ detection and analysis of positive polypeptides were carried out by microarray chip and matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF). The optimal polypeptide sequences were obtained by comparative analysis of multiplex sequences of Clustal X2. Verify the affinity and specificity of peptides at the cellular level. Finally, VEGFR2 targeting drug carrier S1-LSN was prepared by thin-film dispersion method. Human umbilical vein endothelial cells (HUVECs) were used as positive cell models in vitro and mice bearing human colon cancer (HT-29) were used as models to evaluate S1-LS targeting in vitro. Efficiency of drug delivery. Results the microarray chip system captured more than 400 positive peptide beads from the constructed 106 polypeptide library. After sequencing the captured peptide bead -VEGFR2-biotin-streptavidin-magnetic bead bridged magnetic peptide beads, The molecular affinity of VEGFR2 high affinity peptide S1, Leu-Ile-Asn-His-Glu-Trp-LysGlu-Asn-Tyr-Phe-Pro-Leu-Ser-Phe.S1 to VEGFR2 at the molecular level was determined by sequence comparison analysis. A laser confocal microscope was used to locate the S1S1-FITCU labeled by fluorescein isothiocyanate (FITC). It was proved that the overexpression of VEGFR2 was highly efficient in binding with S1. S1 was successfully modified with amphiphilic molecules to obtain uniform size. VEGFR2-targeted liposome S1-LS.S1-LS showed high drug targeting delivery efficiency in vitro positive cells HUVECs and animal tumor model (HT-29 tumor-bearing mice). Conclusion the optimized microarray microarray chip is used for drug delivery. The efficiency of VEGFR2-targeted polypeptide was studied. High throughput screening and identification; After optimizing the polypeptide molecular probes, the specific delivery system for nanopharmaceuticals was successfully constructed. The VEGFR2 targeted peptide S1 modified nano-liposome S1-LS1) successfully realized the highly efficient delivery of tumor targeting drugs S1-LS, which is expected to be used in cancer. In the targeted diagnosis and treatment of the disease, To provide new ideas and methods for accurate diagnosis and treatment.
【学位授予单位】：锦州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R943;R96

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