本文關(guān)鍵詞：側(cè)鏈可還原降解的聚乙烯亞胺衍生物的合成和體外基因釋放　出處：《蘇州大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 基因轉(zhuǎn)染 聚乙烯亞胺 復(fù)合物 質(zhì)粒DNA 生物降解 雙硫鍵

【摘要】：基因治療就是將外源基因引入受感染的細(xì)胞內(nèi)進(jìn)行適當(dāng)?shù)谋磉_(dá),以糾正致病基因所產(chǎn)生的缺陷,從而達(dá)到從根本上治療疾病的目的。 目前實(shí)施基因治療的主要瓶頸是缺乏安全高效的基因傳遞系統(tǒng)。盡管病毒載體具有較高的轉(zhuǎn)染效率,但存在制備復(fù)雜,有安全隱患,包裝容量有限等問題。因此,近年來人們越來越青睞于人工合成的非病毒載體。由于陽離子聚合物作為非病毒性載體有安全性好、制備簡單方便、可多次重復(fù)使用等眾多優(yōu)點(diǎn)而受到研究者們的青睞。其中,聚乙烯亞胺(PEI)因其正電荷密度較高,且可通過“質(zhì)子海綿作用”逃離內(nèi)涵體,以其結(jié)構(gòu)為基礎(chǔ)的新型陽離子聚合物載體成為研究熱點(diǎn)。25 kDa支化PEI是迄今為止轉(zhuǎn)染效率最高的陽離子聚合物載體之一,現(xiàn)已經(jīng)被作為新開發(fā)的體外轉(zhuǎn)染載體的比較標(biāo)準(zhǔn),但其跟病毒性基因載體相比轉(zhuǎn)染效率仍然較低且在體內(nèi)不可降解,細(xì)胞毒性較大。 本論文研究的主要目的在于對轉(zhuǎn)染效果較好的基因載體25 kDa支化PEI進(jìn)行改性,引入在細(xì)胞內(nèi)可還原降解的二硫鍵,制備出新型可降解的聚乙烯亞胺(PEI)衍生物作為基因載體,從而實(shí)現(xiàn)高效低毒的非病毒基因轉(zhuǎn)染。25 kDa支化PEI首先分別與單體Ac-Cys-t~Boc及單體MAc-Cys-t~Boc進(jìn)行邁克爾加成,然后脫保護(hù)形成取代度分別為14、27、34和13、19、38的PEI-Cys衍生物,并分別命名為PEI-(Cys)x(Ac)和PEI-(Cys)x(MAc)。隨后對這些聚合物進(jìn)行了一系列的性能表征。緩沖能力測試表明所有PEI-Cys衍生物的緩沖能力都比25 kDa支化PEI高(21.2-23.1%比15.1%);凝膠電泳結(jié)果表明胱胺的修飾有利于聚合物更好的壓縮DNA,PEI-(Cys)x(Ac)在N/P≥5/1時(shí)可以將DNA壓縮為80-90 nm的小粒子,比聚合物25 kDa支化PEI壓縮的復(fù)合物粒徑小(100-130 nm)。與之對比,聚合物PEI-(Cys)x(MAc)壓縮DNA形成的復(fù)合物粒子則較大(100-180 nm,N/P從30/1到5/1);凝膠電泳及動(dòng)態(tài)光散射(DLS)測試表明PEI-Cys衍生物與DNA形成的復(fù)合物在10 mM二硫蘇糖醇(DTT)的溶液中可以快速地釋放出質(zhì)粒DNA;與25 kDa支化PEI相比所有的PEI-Cys衍生物都具有更低的細(xì)胞毒性(IC50: 100 mg/L versus ca. 11 mg/L);報(bào)告基因pGL3在HeLa細(xì)胞和293T細(xì)胞中的體外基因轉(zhuǎn)染實(shí)驗(yàn)表明,隨著取代度(DS)的提高,PEI-Cys衍生物的轉(zhuǎn)染效率逐漸降低,而且在相似取代度的前提下聚合物PEI-(Cys)_x(MAc)比聚合物PEI-(Cys)_x(Ac)的轉(zhuǎn)染效率高。在無血清和有血清條件下,聚合物PEI-(Cys)14(Ac)及PEI-(Cys)_x(MAc)與DNA在N/P比為10條件下形成的復(fù)合物的轉(zhuǎn)染效率都明顯高于25 kDa支化PEI(最高達(dá)4.1倍)。結(jié)果表明經(jīng)胱胺修飾的可還原降解的新型PEI載體有較好的發(fā)展前景,可望被成功研制為既高效又安全無毒的新一代非病毒性基因轉(zhuǎn)染試劑,應(yīng)用于體內(nèi)外基因轉(zhuǎn)染。
[Abstract]:Gene therapy is to introduce foreign genes into infected cells for proper expression, in order to correct the defects of pathogenic genes, so as to fundamentally cure diseases. At present, the main bottleneck of gene therapy is the lack of a safe and efficient gene transmission system. Although the virus vector has a high transfection efficiency, but the preparation of complex, there are hidden dangers of safety. Packaging capacity is limited. Therefore, in recent years, people are more and more in favor of synthetic non-viral carriers. As cationic polymers as non-viral carriers have good security, simple and convenient preparation. Polyethyleneimide (PEI) is favored by researchers because of its high positive charge density, and it can escape from the connotation by "proton sponge action". The novel cationic polymer carrier based on its structure has become a hot topic. 25 kDa branched PEI is one of the most efficient cationic polymer carriers so far. Now it has been used as the comparative standard of the newly developed in vitro transfection vector, but its transfection efficiency is still lower than that of viral gene vector, which is not degradable in vivo and has greater cytotoxicity. The main purpose of this study was to modify the transfected gene vector 25 kDa branched PEI and introduce the reductive and degradable disulfide bonds in the cells. A novel biodegradable PEI derivative was prepared as a gene carrier. So as to achieve high efficiency and low toxicity of non-viral gene transfection. 25. KDa branched PEI was firstly added to monomer Ac-Cys-t~Boc and monomeric MAc-Cys-t~Boc respectively. Then the PEI-Cys derivatives with the degree of substitution of 14 ~ (27) ~ (34) and 13 ~ (19) ~ (38) were formed by deprotection. They were named PEI-CysCysX Acand (PEI-CysCysX) and PEI-CysCysTX (MAcl), respectively. The buffering ability of all PEI-Cys derivatives was higher than that of 25 kDa branched PEI. 21.2-23.1% vs 15.1%; The results of gel electrophoresis showed that the modification of cysteamine was beneficial to the better compression of DNA. DNA can be compressed to 80-90 nm particles at N / P 鈮，

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