本文選題：無規(guī)線團(tuán) + 膠束結(jié)構(gòu)��； 參考：《天津大學(xué)》2015年碩士論文

【摘要】：基因治療有望成為治療癌癥等疾病的有效方法,基因治療獲得臨床應(yīng)用的關(guān)鍵是高效遞送基因藥物的載體材料。作為非病毒基因載體的一種,聚陽離子能夠有效的避免酶解并促進(jìn)細(xì)胞攝取。雖然許多聚陽離子在很多方面表現(xiàn)出較低的免疫原性、制備簡單以及成本低等優(yōu)點(diǎn),但是較低的基因轉(zhuǎn)染效率仍然是基因治療的瓶頸。為提高基因遞送效率,采取了許多種化學(xué)修飾的方法。本研究我們利用PVP具有協(xié)同遞送基因藥物的性能及PDMAEMA的遞送基因藥物的優(yōu)良性能制備具有不同PVP鏈長的雙親水性聚合物PVP-g-PDMAEMA。我們研究了親水鏈長度對基因遞送的影響,優(yōu)化親水端長度。在此基礎(chǔ)上制備兩親性聚合物——PVP-g-PDMAEMA-b-PMMA。探究了以PVP-g-PDMAEMA形成的無規(guī)線團(tuán)結(jié)構(gòu)和PVP-g-PDMAEMA-b-PMMA形成的膠束結(jié)構(gòu)對基因遞送的影響。PVP-g-PDMAEMA-b-PMMA具有較低的CMC值,約為6.3×10-3 mg/mL。PVP-g-PDMAEMA-b-PMMA/pDNA形成的納米粒粒徑與電位均比PVP-g-PDMAEMA/pDNA明顯小。與PVP-g-PDMAEMA相比,PVP-g-PDMAEMA-b-PMMA顯示出較好的基因轉(zhuǎn)染效率,在293T細(xì)胞上以較低的N/P比約為3:1時(shí)明顯的超過PEI的轉(zhuǎn)染效率。因此,結(jié)果表明PVP-g-PDMAEMA-b-PMMA是一種高效的基因載體。聚陽離子雖然體外具有較高的轉(zhuǎn)染效率,但體內(nèi)轉(zhuǎn)染效率較低。為了解決此問題,我們發(fā)展了基于BSA的電荷反轉(zhuǎn)和二硫鍵可逆形成斷裂的方法。采用巰基乙胺修飾的牛血清白蛋白(SH-BSA)與PVP-g-PDMAEMA-b-PMMA/pDNA通過靜電引力結(jié)合,形成三元復(fù)合物,且SH-BSA表面的巰基氧化交聯(lián)成二硫鍵,提高其穩(wěn)定性。SH-BSA的等電點(diǎn)從4.7提高到5.3。在生理?xiàng)l件下,SH-BSA帶有負(fù)電荷;在pH為5.0時(shí),SH-BSA帶有正電荷。SH-BSA隨著pH的變化發(fā)生電荷反轉(zhuǎn)作用,與PVP-g-PDMAEMA-b-PMMA/pDNA由靜電引力變成靜電斥力,但由于交聯(lián)二硫鍵的存在,SH-BSA無法從PVP-g-PDMAEMA-b-PMMA/pDNA表面分離。當(dāng)加入谷胱甘肽后,二硫鍵斷裂,SH-BSA從PVP-g-PDMAEMA-b-PMMA/pDNA分離。所以,具有適當(dāng)穩(wěn)定性的SH-BSA有利于基因材料的轉(zhuǎn)移和釋放。
[Abstract]:Gene therapy is expected to be an effective method for the treatment of cancer and other diseases. The key to the clinical application of gene therapy is the efficient delivery of carrier materials for gene drugs. As a non-viral gene vector, polycations can effectively avoid enzymatic hydrolysis and promote cell uptake. Although many polycations have the advantages of low immunogenicity, simple preparation and low cost in many aspects, the low efficiency of gene transfection is still the bottleneck of gene therapy. In order to improve the efficiency of gene delivery, many methods of chemical modification have been adopted. In this study, we prepared PVP-g-PDMAEMA-a double hydrophilic polymer with different PVP chain lengths by using PVP as a synergistic delivery gene drug and PDMAEMA as a delivery gene drug. We studied the effect of hydrophilic chain length on gene delivery and optimized the hydrophilic end length. On this basis, amphiphilic polymer PVP-g-PDMAEMA-b-PMMA was prepared. The effect of random coil structure formed by PVP-g-PDMAEMA and micelle structure formed by PVP-g-PDMAEMA-b-PMMA on gene delivery. PVP-g-PDMAEMA-b-PMMA has a lower CMC value. The particle size and potential of the nanoparticles formed by about 6.3 脳 10-3 mg/mL.PVP-g-PDMAEMA-b-PMMA/pDNA are obviously smaller than that of PVP-g-PDMAEMA/pDNA. Compared with PVP-g-PDMAEMA, PVP-g-PDMAEMA-b-PMMA showed better gene transfection efficiency, and the transfection efficiency of PEI on 293T cells was significantly higher than that of PEI when the ratio of N / P was about 3:1. Therefore, the results show that PVP-g-PDMAEMA-b-PMMA is an efficient gene vector. Although polycation has high transfection efficiency in vitro, in vivo transfection efficiency is low. In order to solve this problem, we developed a method of charge inversion and reversible fracture of disulfide bond based on BSA. Bovine serum albumin (SH-BSA) modified by mercaptoethylamine was combined with PVP-g-PDMAEMA-b-PMMA/pDNA by electrostatic force to form a ternary complex, and the sulfhydryl group on the surface of SH-BSA was crosslinked to form disulfide bond. The isoelectric point of SH-BSA was increased from 4.7 to 5.3. Under physiological conditions, SH-BSA has negative charge, and when pH is 5.0, SH-BSA has positive charge .SH-BSA changes with pH, and changes from electrostatic gravity to electrostatic repulsion with PVP-g-PDMAEMA-b-PMMA/pDNA, but SH-BSA can not be separated from PVP-g-PDMAEMA-b-PMMA/pDNA surface because of the existence of cross-linked disulfide bond. When glutathione was added, the disulfide bond was separated from PVP-g-PDMAEMA-b-PMMA/pDNA. Therefore, SH-BSA with proper stability is conducive to the transfer and release of genetic materials.
【學(xué)位授予單位】：天津大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R450;O631

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