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  本文選題：多肽探針　切入點：VEGFR2　出處：《錦州醫(yī)科大學》2017年碩士論文　論文類型：學位論文

【摘要】：目的化療是目前癌癥治療中常用的手段,但傳統(tǒng)的化療藥物仍面臨著許多亟待解決的問題,如體內生物利用度低、溶解性差、嚴重的不良副作用以及易產生耐藥性等。近年來,將納米技術應用于癌癥的治療在一定程度上增加了化療藥物的療效,但是仍然存在一些不足,如藥物不可控的釋放、腫瘤病灶靶向性低等。然而,功能性靶向藥物遞送系統(tǒng)可以有效的解決常規(guī)納米藥物載體存在的一些問題,如在一個納米體系中可實現(xiàn)高效的腫瘤靶向、藥物控制釋放等多種功能。為此,我們構建了一種新穎的靶向新生血管生成的抗腫瘤納米藥物載體(S1-LS),旨在提高小分子化療藥物的抗腫瘤療效,同時也能夠減小藥物對其他正常組織造成的毒副作用。方法針對血管內皮細胞生長因子受體2(VEGFR2),通過多肽固相合成技術、利用混合均分法構建“一珠一物”(OBOC)多肽文庫。隨后,利用免疫磁珠的方法篩選陽性肽珠,同時通過微陣列芯片結合基質輔助激光解吸電離飛行時間質譜(MALDI-TOF MS)對陽性多肽進行原位檢測分析,Clustal X2多重序列對比分析篩選獲得最優(yōu)多肽序列。分別在分子和細胞水平上驗證多肽的親和性和特異性。最后,采用薄膜分散法制備VEGFR2靶向納米藥物載體(S1-LS),并在體外細胞水平以人臍靜脈血管內皮細胞(HUVEC)為陽性細胞模型以及動物水平以人結腸癌(HT-29)荷瘤小鼠為模型評價S1-LS靶向遞送藥物的效率。結果微陣列芯片系統(tǒng)從所構建106多肽文庫中捕獲400多個陽性肽珠。原位裂解、測序被捕獲的肽珠-VEGFR2-生物素-鏈霉親和素-磁珠橋連的磁性肽珠后,通過序列對比分析篩選獲得VEGFR2高親和性多肽S1(Leu-Ile-Asn-His-Glu-Trp-LysGlu-Asn-Tyr-Phe-Pro-Leu-Ser-Phe)。S1與VEGFR2在分子水平的親和性由表面等離子共振成像系統(tǒng)(SPRi)測得(KD=1.31×10-7 M)。在細胞水平上,通過激光共聚焦顯微鏡定位異硫氰酸熒光素(FITC)標記的S1(S1-FITC),證明了VEGFR2過表達的陽性細胞(HUVEC)與S1具有高的結合效率。S1成功修飾兩親性分子后制備得到大小均一、分散均勻的VEGFR2-靶向納米脂質體S1-LS。S1-LS在體外陽性細胞(HUVEC)及體內動物腫瘤模型(HT-29荷瘤小鼠)的實驗中都顯示了較高的藥物靶向遞送效率。結論利用優(yōu)化的微陣列芯片成功進行了VEGFR2-靶向多肽的高效、高通量的篩選和鑒定;且在優(yōu)化多肽分子探針后成功構建了特異性靶向納米藥物遞送系統(tǒng)(S1-LS)�；赩EGFR2靶向多肽S1修飾的納米脂質體(S1-LS)成功實現(xiàn)了高效的腫瘤靶向藥物遞送。S1-LS有望被用于癌癥的靶向診斷和治療中,為實現(xiàn)精準化診斷和治療提供新思路、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.
【學位授予單位】：錦州醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R943;R96

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本文編號：1599420