本文選題：基因治療 + 超分子組裝　； 參考：《浙江大學(xué)》2007年博士論文

【摘要】： 現(xiàn)代基因技術(shù)和人類基因組工程圖譜的完成為采用基因分子生物學(xué)方法治療各類疾病，提高人類生命質(zhì)量提供了廣闊的前景�；蛑委煹难芯恳呀�(jīng)深入醫(yī)學(xué)研究的各個領(lǐng)域，成為當(dāng)今最活躍的生物高技術(shù)領(lǐng)域之一。采用超分子組裝技術(shù)制備的非病毒基因傳遞體系，由于具有合適的納米尺寸、可控的結(jié)構(gòu)及良好的生物相容性，顯示出巨大的發(fā)展?jié)摿皯?yīng)用前景。如何提高非病毒基因載體在體內(nèi)的穩(wěn)定傳遞和高效轉(zhuǎn)染是這類傳遞體系所需解決的關(guān)鍵的科學(xué)問題。病毒是由蛋白質(zhì)殼層所包被的具有核殼結(jié)構(gòu)的天然超分子組裝體，其細(xì)胞外穩(wěn)定存在、細(xì)胞內(nèi)解組裝響應(yīng)并高效轉(zhuǎn)染的特點(diǎn)為非病毒基因傳遞體系的設(shè)計提供了很好的啟迪。本論文以構(gòu)建穩(wěn)定傳遞和高效轉(zhuǎn)染的非病毒基因傳遞體系為研究背景，采用超分子組裝技術(shù)與基因技術(shù)相結(jié)合，制備了模擬病毒結(jié)構(gòu)特點(diǎn)的非病毒基因超分子組裝體，初步探討了超分子組裝體的結(jié)構(gòu)與基因轉(zhuǎn)染效率的內(nèi)在聯(lián)系。 聚乙烯亞胺(PEI)具有的“質(zhì)子海綿效應(yīng)”有利于其從溶酶體中逃離出來，是目前常用的一類非病毒基因載體。但PEI／DNA組裝體在生理鹽溶液中不穩(wěn)定、易聚集，制備PEG化的基因超分子組裝體可解決上述問題。本文首先通過含PEG鏈段的兩親聚合物的自組織共混改性，，采用超分子技術(shù)構(gòu)建了基于疏水作用力的新型PEG化基因超分子組裝體。膽固醇—聚乙二醇(CPEG)的加入用量及次序?qū)虺肿咏M裝體在生理鹽溶液中的穩(wěn)定性產(chǎn)生很大影響。將CPEG與PEI_(25k)的混合溶液加入到等體積的DNA溶液中，制備的PEI_(25k)／CPEG／DNA組裝體在生理鹽溶液中的穩(wěn)定性得到很大提高；而在PEI_(25k)／DNA組裝體形成之后，再加入CPEG則不能提高其穩(wěn)定性。這一現(xiàn)象顯示新型PEG化基因組裝體的形成，與疏水膽固醇參與到疏水核(由PEI_(25k)和DNA靜電中和形成)的組裝密切相關(guān)。研究進(jìn)一步采用以表達(dá)綠色熒光蛋白的質(zhì)粒pEGFP為模型，將制備的基因超分子組裝體在生理鹽溶液中放置不同的時間，并轉(zhuǎn)染HEK293T細(xì)胞。熒光顯微鏡及流式細(xì)胞儀結(jié)果表明：隨著放置時間的延長，PEI_(25k)／DNA組裝體的轉(zhuǎn)染效率顯著降低；而通過CPEG共混改性后的PEI_(25k)／CPEG／DNA基因組裝體由于在生理鹽溶液中的穩(wěn)定性得到很大提高，合適的粒子尺寸使其在體外的基因轉(zhuǎn)染效率顯著提高。含PEG鏈段兩親分子的自組織共混為制備具有新型的PEG化基因超分子組裝體，提高其在生理鹽溶液中的穩(wěn)定性及基因轉(zhuǎn)染效率，提供了簡單有效的新途徑。 針對基因載體與DNA分子在傳遞過程中的穩(wěn)定締合和在細(xì)胞內(nèi)的解離釋放這一對矛盾，本研究依據(jù)病毒的蛋白質(zhì)殼層細(xì)胞內(nèi)外響應(yīng)傳遞的特點(diǎn)，開展了生物響應(yīng)型非病毒基因傳遞體系的研究。研究利用細(xì)胞內(nèi)高濃度谷胱甘肽對-S-S-鍵的響應(yīng)特性，由低分子量PEI_(1800)出發(fā)，通過與二甲基3，3’-二硫代-雙(丙亞氨酸酯)二氯化氫(DTBP)的交聯(lián)反應(yīng)，合成了一系列含-S-S-鍵的交聯(lián)聚乙烯亞胺(CLPEI)，通過對交聯(lián)程度和組裝條件的調(diào)控，成功制備了谷胱甘肽響應(yīng)的CLPEI_(50％)／DNA基因超分子組裝體。實(shí)驗(yàn)結(jié)果表明：CLPEI_(50％)具有最佳的締合DNA分子的能力，并在pH值為7.4到5的范圍內(nèi)，依然具有足夠的質(zhì)子緩沖能力，有利于基因超分子組裝體從溶酶體中逃離出來。在pH為6.0、NaCl濃度為20mM的條件下，能與DNA形成直徑在150 nm左右的球形粒子。在模擬細(xì)胞內(nèi)谷胱甘肽濃度的條件下，CLPEI_(50％)分子中-S-S-鍵的斷裂導(dǎo)致高分子量聚陽離子轉(zhuǎn)變?yōu)榈头肿恿科瑪�，�?shí)現(xiàn)組裝體的解組裝響應(yīng)。體外細(xì)胞培養(yǎng)結(jié)果表明：這種仿生交聯(lián)的基因組裝體可顯著降低細(xì)胞毒性，并有效轉(zhuǎn)染細(xì)胞。 為進(jìn)一步增強(qiáng)基因超分子組裝體的穩(wěn)定性和轉(zhuǎn)染效率，研究以病毒組裝過程中生物大分子交聯(lián)的“籠狀”結(jié)構(gòu)為啟示，開展了“籠狀”仿生交聯(lián)組裝體的研究。合成了含巰基的聚乙烯亞胺(HS-PEI)，與DNA組裝后，分別通過空氣中巰基的氧化交聯(lián)制備殼層原位交聯(lián)的基因超分子組裝體；通過金納米粒子(AuNP)與巰基的共價締合，制備殼層納米金交聯(lián)的組裝體。熒光光譜測定結(jié)果表明：在N／P值為10的條件下，巰基化聚乙烯亞胺(HS-PEI)能有效誘導(dǎo)DNA分子的締合。在pH為6.0、NaCl濃度為20 mM的HEPEs緩沖溶液中，這種殼層交聯(lián)的組裝體的尺寸在150 nm左右。對于殼層AuNP交聯(lián)的Au-S-PEI／DNA組裝體，由TEM圖片我們可清晰觀察到殼層金納米粒子的存在，且隨著AuNP加入量的增加，殼層交聯(lián)的金納米粒子含量也顯著增加。與殼層未交聯(lián)的基因超分子組裝體相比，殼層交聯(lián)的“籠狀”結(jié)構(gòu)可有效提高組裝體在生理鹽溶液中的穩(wěn)定性，并顯示出很好的谷胱甘肽響應(yīng)特性。體外細(xì)胞培養(yǎng)結(jié)果表明：通過選擇合適的組裝條件，仿生交聯(lián)的“籠狀”非病毒基因傳遞體系可有效轉(zhuǎn)染。 采用超分子組裝技術(shù)構(gòu)建仿病毒基因傳遞體系，使其既具備非病毒載體的低毒性、低免疫原性的特點(diǎn)，又具有病毒載體高效轉(zhuǎn)染的特性，將是未來基因傳遞體系的發(fā)展方向。本論文采用超分子組裝手段，通過含PEG鏈段兩親聚合物的 自組織共混改性，制備了新型PEG化基因超分子組裝體；利用谷胱甘肽的生物開關(guān)效應(yīng)，設(shè)計了含二硫鍵的聚陽離子，制備了細(xì)胞內(nèi)外響應(yīng)傳遞的非病毒基因傳遞體系；通過制備巰基化聚乙烯亞胺，構(gòu)建了殼層原位交聯(lián)和納米金交聯(lián)的“籠狀”基因超分子組裝體，為新型仿病毒基因傳遞體系的設(shè)計提供了切實(shí)可行的途徑，并在基因治療領(lǐng)域顯示出廣闊的應(yīng)用發(fā)展前景。
[Abstract]:The completion of modern gene technology and human genome engineering map provides a broad prospect for the use of gene molecular biology to treat various diseases and improve the quality of human life. The research of gene therapy has already deepened every field of medical research and became one of the most active fields of biotechnology. The non viral gene transfer system, due to the proper nanoscale size, controllable structure and good biocompatibility, shows great potential for development and application prospects. How to improve the stable transmission and efficient transfection of non viral vector in vivo is the key scientific problem to be solved in this kind of transmission system. It is a natural supramolecular assembly with nuclear shell structure covered by protein shell, and its extracellular stability exists. The characteristics of intracellular solution assembly response and efficient transfection provide a good inspiration for the design of non viral gene transfer system. This paper is to construct a non viral gene transfer system for stable transmission and efficient transfection. In the context of the combination of supramolecular assembly and gene technology, a non viral gene supramolecular assembly that mimic the structural characteristics of the virus was prepared. The intrinsic relationship between the structure of supramolecular assembly and gene transfection efficiency was preliminarily discussed.
The "proton sponge effect" of polyethyleneimine (PEI) is beneficial to its escape from the lysosome. It is a commonly used non viral gene carrier. However, the PEI / DNA assembly is unstable in the physiological salt solution and is easy to gather. The preparation of PEG gene supramolecular assembly can solve the above problems. First of all, two of the PEG segments are used. A novel PEG gene supramolecular assembly based on hydrophobic interaction was constructed by the self-organized blending modification of the amphiphilic polymer. The dosage and order of the addition of cholesterol and polyethylene glycol (CPEG) had a great effect on the stability of the gene supramolecular assembly in the physiological salt solution. The combination of CPEG and PEI_ (25K) mixed solution was added. In the DNA solution of equal volume, the stability of the prepared PEI_ (25K) / CPEG / DNA assembly in the physiological salt solution is greatly improved, and the addition of CPEG after the formation of PEI_ (25K) / DNA assembly can not improve its stability. This phenomenon shows that the new PEG matrix is formed by the formation of the assembly and hydrophobic cholesterol is involved in hydrophobicity. Nucleation is closely related to the assembly of PEI_ (25K) and DNA electrostatic neutralization. The study further uses plasmid pEGFP expressing green fluorescent protein as a model to place the prepared gene supramolecular assembly in a physiological salt solution for different time and transfect HEK293T cells. The results of fluorescence microscopy and flow cytometry show that with the placement of the fluorescent microscope and flow cytometry The transfection efficiency of the PEI_ (25K) / DNA assembly was significantly reduced, while the PEI_ (25K) / CPEG / DNA genomic assembly modified by CPEG was greatly improved because of the stability in the physiological salt solution. The appropriate particle size made the gene transfection efficiency in the vitro significantly improved. The self group of the two parent molecules containing PEG chain segments was improved. In order to prepare a new PEG gene supramolecular assembly and improve its stability in physiological salt solution and gene transfection efficiency, a new and effective new way is provided.
In view of the contradiction between the stable association of gene carriers and DNA molecules during the transfer process and the release of intracellular release, this study carried out a study on the biological response non viral gene transfer system based on the characteristics of the response transmission of the protein shell inside and outside of the virus, and studied the use of high concentration of intracellular glutathione to the -S-S- key. A series of crosslinked polyethylenimines (CLPEI) containing -S-S- bonds were synthesized by crosslinking reaction with two methyl 3,3 '- two thiosulfate (malonic acid) two hydrogen chloride (DTBP) by the response characteristic of low molecular weight PEI_ (1800). The CLPEI_ (50%) / DNA base response of glutathione response was successfully prepared by the regulation of the crosslinking degree and the assembly conditions. The results of the supramolecular assembly show that CLPEI_ (50%) has the best ability to associate DNA molecules, and still has enough proton buffering ability in the range of 7.4 to 5 of pH, which is beneficial to the escape of the gene supramolecular assembly from the lysosome. Under the condition of pH and 20mM, the diameter of DNA can be 15. 0 nm spherical particles. Under the condition of simulated cell Uchiya Ka concentration, the fracture of the -S-S- bond in the CLPEI_ (50%) molecule leads to the transformation of high molecular weight polycation to a low molecular weight fragment and the assembly response to the assembly. The results of cell culture in vitro show that this crosslinked genome assembly can significantly reduce cytotoxicity. Sex, and transfection of cells effectively.
In order to further enhance the stability and transfection efficiency of gene supramolecular assembly, the study of "cage like" bionic crosslinking assembly was carried out by the "cage like" structure of biological macromolecule crosslinking in the process of virus assembly. The polyethylenimide (HS-PEI) containing sulfhydryl group was synthesized. After assembling with DNA, the sulfhydryl oxygen in the air was carried out respectively. The in-situ crosslinked gene supramolecular assembly of shell was prepared by chemical crosslinking; the shell nanoscale crosslinked assembly was prepared by covalent Association of gold nanoparticles (AuNP) with the sulfhydryl group. The results of fluorescence spectrometry showed that under the condition of N / P 10, mercapto polyethyleneimine (HS-PEI) could effectively induce the association of DNA molecules. In pH, 6, NaCl In the HEPEs buffer solution with a concentration of 20 mM, the size of the shell crosslinked assembly is about 150 nm. For the AuNP crosslinked Au-S-PEI / DNA assembly of the shell layer, the existence of the shell gold nanoparticles can be clearly observed from the TEM picture. With the increase of AuNP addition, the content of the gold nanoparticles crosslinked by the shell is also significantly increased. Compared with the non crosslinked gene supramolecular assembly, the shell crosslinked "cage" structure can effectively improve the stability of the assembly in the physiological salt solution, and show a good response characteristic of glutathione. In vitro cell culture results show that the "cage like" non viral gene transfer through the selection of suitable assembly conditions and bionic crosslinking The system can be transfected effectively.
Using supramolecular assembly technology to construct a virus gene transfer system, which not only has the characteristics of low toxicity, low immunogenicity, and high efficiency of transfection of virus carriers, which will be the development direction of the gene delivery system in the future. This paper uses the supramolecular assembly method, through the two amphiphilic polymer containing PEG segment.
A novel PEG gene supramolecular assembly was prepared by self tissue blending, and a polycation containing two sulfur bonds was designed by using the biological switching effect of glutathione, and the non viral gene transfer system was prepared in vitro and in vitro. By preparing mercapto polyethyleneimine, the shell in situ crosslinking and nanoscale crosslinking were constructed. The "cage" gene supramolecular assembly provides a practical way for the design of a new type of virus like gene transfer system, and has shown a broad prospect of application in the field of gene therapy.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2007
【分類號】：R346

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 汪存東;;超分子科學(xué)中的仿生自組裝[J];現(xiàn)代化工;2008年07期

相關(guān)博士學(xué)位論文 前1條

1 張鋒;鈦基種植體表面仿細(xì)胞外基質(zhì)活性涂層的設(shè)計、構(gòu)建及其生物學(xué)評價[D];浙江大學(xué);2010年

本文編號：2011633