本文選題：人血清白蛋白　切入點：黃酮衍生物　出處：《清華大學》2014年碩士論文　論文類型：學位論文

【摘要】：人血清白蛋白（HSA）是一個研究藥物與蛋白質(zhì)相互作用的模式蛋白。在本研究中，我們對實驗室設計合成的化合物3d（對肝癌類細胞HepG2有潛在抗增值活性）與HSA的相互作用進行了探索。研究中使用熒光光譜法、紫外-可見光光譜法、圓二色譜法和分子模擬手段對二者相互作用情況進行表征。熒光光譜法實驗結果表明HSA的熒光發(fā)射可以被3d明顯的淬滅，并且最大發(fā)射波長從360nm紅移到了363nm。利用Stern-volmere方程和修飾的Stern-volmere方程計算可知3d對HSA的熒光淬滅屬于靜態(tài)淬滅，二者在298K時的結合常數(shù)為5.26±0.04×104L mol-1。經(jīng)過計算相關熱力學參數(shù)得知，靜電作用力在3d-HSA復合物穩(wěn)定中起到了主要作用。從紫外-可見光光譜和圓二色譜結果都可以看出，HSA在與3d結合之后構象發(fā)生了改變，而且隨著3d濃度的加大HSA結構變得松散、α-螺旋含量略有降低。分子模擬結果顯示化合物3d可以恰好進入HSA的一個亞結構域IIA，而這個區(qū)域與HSA的儲存和轉運功能相關。該區(qū)域周邊氨基酸殘基ARG218、ARG222和LYS199與3d相應基團形成了三個正離子-π相互作用以及三個氫鍵作用�？傊�，黃酮類衍生物3d可以與HSA以非共價鍵方式中等結合力相結合，這樣它就可以被HSA在血液中進行轉運。 另外還利用SELEX技術結合RFD分子模型對乳腺癌細胞MCF7進行了適配體的篩選工作。研究中使用MCF7細胞的培養(yǎng)液作為篩選的對象，根據(jù)細胞外分泌物的不同區(qū)分不同種類的癌細胞。通過24輪重復篩選和富集，最終得到了與MCF7細胞外液反應的分子庫�？寺『蜏y序之后獲得了一些確定寡核苷酸序列，對其中的某些序列研究顯示，它們均可以與MCF7的外液進行反應，獲得明顯的熒光信號。對它們細胞識別特異性研究發(fā)現(xiàn)不僅可以識別MCF7細胞，還對兩種肝源的細胞HepG2和HL7702有較強的反應。另外對MCF10A的反應信號也較強，說明在這幾種細胞的分泌物中都具有某一種或一類分子，，而這些分子恰好被篩選到的適配體分子庫所識別。對序列ABE2-1細胞靈敏度檢測實驗表明，其最低檢測細胞濃度可以達到103細胞/mL。采用超濾膜對細胞外液進行分段的方式初步確定靶標的分子量范圍,最終確定靶標分子量范圍在30-50kD之間。
[Abstract]:Human Serum Albumin (HSA) is a model protein that studies the interaction between drugs and proteins. We have explored the interaction of compounds designed and synthesized in laboratory for 3 d (with potential antiproliferative activity to hepatoma cell HepG2) with HSA. The fluorescence spectroscopy, UV-Vis spectroscopy, UV-Vis spectroscopy were used in the study. The interaction between the two was characterized by circular dichroism and molecular simulation. The fluorescence spectra showed that the fluorescence emission of HSA could be quenched by 3 d. The maximum emission wavelength was shifted from 360 nm to 363nm.The fluorescence quenching of HSA at 298K was statically quenched by Stern-volmere equation and modified Stern-volmere equation. The binding constant was 5.26 鹵0.04 脳 10 4 L mol -1. Electrostatic force plays an important role in the stabilization of 3d-HSA complex. The conformation of HSA has changed after 3 d binding from UV-Vis spectra and circular dichroism spectra. Moreover, with the increase of 3D concentration of HSA, the content of 偽 -helix decreases slightly. The molecular simulation results show that the compound 3D can enter a subdomain of HSA, which is related to the storage and transport function of HSA. The amino acid residues ARG218N ARG222 and LYS199 formed three positive ion-蟺 interactions and three hydrogen bonds with their 3D corresponding groups. The flavonoid derivatives can bind to HSA in a non-covalent bond form for 3 d, so that it can be transported by HSA in the blood. In addition, the aptamer of breast cancer cell MCF7 was screened by SELEX technique and RFD molecular model. The culture medium of MCF7 cells was used as the screening object. According to different extracellular secretions, different types of cancer cells were differentiated. After 24 rounds of repeated screening and enrichment, a molecular library of extracellular fluid reaction with MCF7 was obtained. After cloning and sequencing, some definite oligonucleotide sequences were obtained. Studies on some of the sequences showed that they could react with the external fluid of MCF7 to obtain obvious fluorescence signals. The recognition specificity of their cells was found to recognize not only MCF7 cells, but also MCF7 cells. There was also a strong response to HepG2 and HL7702 in two liver-derived cells, and a stronger signal to MCF10A, indicating that there was one or a class of molecules in the secretions of these cells. These molecules were identified by a library of aptamer molecules that were screened. Sensitivity tests for sequenced ABE2-1 cells showed that, The lowest cell concentration can reach 103 cell / mL. The molecular weight range of target is determined by using ultrafiltration membrane to segment the extracellular fluid, and the molecular weight range of target is between 30 and 50 KD.
【學位授予單位】：清華大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R96;O657.3

【參考文獻】

相關期刊論文 前1條

1 Charles S. Buer;Nijat Imin;Michael A. Djordjevic;;Flavonoids:New Roles for Old Molecules[J];Journal of Integrative Plant Biology;2010年01期

本文編號：1628425