【摘要】：隨著癌癥以及其它基因類疾病發(fā)病率的升高,基因治療在癌癥以及其它疾病治療中的應(yīng)用引起人們的廣泛關(guān)注�；蛑委�(gene therapy)是指將外源正�；�?qū)氚屑?xì)胞,以糾正或補(bǔ)償因基因缺陷和異常引起的疾病,以達(dá)到治療目的的一種治療手段�；蛑委煹年P(guān)鍵在于通過安全、高效的載體將治療基因傳遞到靶細(xì)胞�；騻鬟f常用的載體一般分為病毒載體和非病毒載體。雖然病毒載體的基因傳遞效率高,但它的免疫源性、致癌性和細(xì)胞毒性較高等缺陷限制了其在體內(nèi)基因治療中的應(yīng)用。近年來,研究者開始致力于探索非病毒載體在基因治療中的應(yīng)用潛能。 在本研究中,我們報(bào)道了兩類基于第五代樹狀大分子(G5.NH2)的新型非病毒基因傳遞載體,分別是乙�；揎椀牡谖宕鷺錉畲蠓肿影{米金顆粒(Au DENPs-Ac)和聚乙二醇修飾的第五代樹狀大分子包裹納米金顆粒(Au DENPs-PEG)。通過核磁、紫外分光光度計(jì)以及透射電鏡對(duì)材料的物理化學(xué)特征進(jìn)行表征；瓊脂糖凝膠電泳技術(shù)確定載體完全結(jié)合基因的N/P；水動(dòng)力粒徑大小和電勢(shì)正負(fù)的測(cè)量初步研究在不同的N/P下,載體／基因是否形成了有利于進(jìn)入細(xì)胞的復(fù)合物。綠色熒光蛋白和熒光素酶的表達(dá)用來研究載體傳遞質(zhì)粒DNA的效率；Western blot技術(shù)用來研究載體傳遞Bcl-2-siRNA的效率；能夠持續(xù)表達(dá)EGFP的Hela細(xì)胞用來研究材料傳遞ssDNA的效率。共聚焦顯微鏡技術(shù)以及流式細(xì)胞技術(shù)也被用來研究載體／基因復(fù)合物的細(xì)胞內(nèi)吞效率。 結(jié)果表明：Au DENPs-Ac能夠顯著地降低Au DENPs的細(xì)胞生物學(xué)毒性,但不影響Au DENPs的基因傳遞效率。Au DENPs-PEG不僅能高效地壓縮pDNA、siRNA和ssDNA形成有利于基因傳遞的復(fù)合物,而且顯著地提高了GS.NH2的基因傳遞效率。在氯金酸與樹狀大分子比相同的條件下,低分子量的聚乙二醇(mPEG,分子量為2000)修飾的Au DENPs比高分子量的mPEG(分子量為5000)修飾的Au DENPs傳遞質(zhì)粒DNA的效率高。同時(shí),在N/P=5時(shí),分子量為2000的mPEG修飾的AuDENPs (Au0)50-G5.NH2-mPEG2k,0)表現(xiàn)出最高的傳遞siRNA和質(zhì)粒DNA的效率。同時(shí),Au DENPs-PEG在傳遞ssDNA方面也有潛在的應(yīng)用價(jià)值。上述研究為功能化樹狀大分子包金材料在基因傳遞領(lǐng)域的應(yīng)用奠定了基礎(chǔ)。
[Abstract]:With the increasing incidence of cancer and other genetic diseases, the application of gene therapy in the treatment of cancer and other diseases has attracted wide attention. Gene therapy (gene therapy) is a therapeutic method that inducts exogenous normal genes into target cells to correct or compensate for diseases caused by gene defects and abnormalities. The key to gene therapy is to transfer therapeutic genes to target cells through safe and efficient vectors. The vectors commonly used for gene transmission are generally divided into viral vectors and non-viral vectors. Although the gene transfer efficiency of virus vector is high, its defects such as immunogenicity, carcinogenicity and cytotoxicity limit its application in gene therapy in vivo. In recent years, researchers have begun to explore the potential of non-viral vectors in gene therapy. In this study, we reported two novel non-viral gene transfer vectors based on the fifth generation dendritic macromolecule (G5.NH2). The fifth generation dendrimer coated gold nanoparticles (au DENPs-Ac) were modified by acetyl group and the au DENPs-PEG particles (au DENPs-PEG) by polyethylene glycol modified dendrimer nanoparticles. The physical and chemical characteristics of the material were characterized by NMR, UV spectrophotometer and transmission electron microscope. Preliminary study on the measurement of hydrodynamic particle size and potential positive or negative whether the vector / gene formed a complex favourable to cell entry under different N / P conditions. The expression of green fluorescent protein and luciferase was used to study the efficiency of vector transfer plasmid DNA. Western blot technique was used to study the efficiency of vector delivery of Bcl-2-siRNA, and Hela cells, which could continuously express EGFP, were used to study the efficiency of ssDNA transfer. Confocal microscopy and flow cytometry have also been used to study the endocytosis efficiency of vector / gene complexes. The results showed that DENPs-Ac could significantly reduce the cytotoxicity of au DENPs, but did not affect the gene transfer efficiency of au DENPs. Au DENPs-PEG could not only efficiently compress DENPs siRNA and ssDNA to form a gene transfer complex. Moreover, the gene transmission efficiency of GS.NH2 was improved significantly. Under the same ratio of chloruronic acid to dendritic macromolecule, au DENPs modified with low molecular weight polyethylene glycol (mPEG, molecular weight 2000) is more efficient than au DENPs transfer plasmid DNA modified by high molecular weight mPEG (molecular weight 5000). At N / P = 5, mPEG modified AuDENPs (Au0) 50-G5.NH2-mPEG2kU _ 0 with molecular weight of 2000 exhibited the highest transfer efficiency of siRNA and plasmid DNA. At the same time, au DENPs-PEG also has potential application value in ssDNA transmission. These studies have laid a foundation for the application of functional dendritic macromolecular gold inclusion materials in gene transfer.
【學(xué)位授予單位】：東華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R943

【共引文獻(xiàn)】

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4 ;Poly(ethylenimine)-grafted-poly[(aspartic acid)-co-lysine]: A Non-viral Polymer with Potential for DNA Delivery[J];Chinese Chemical Letters;2004年01期

5 王幽香,沈家驄;谷胱甘肽響應(yīng)的非病毒基因傳遞體系研究[J];高等學(xué)�；瘜W(xué)學(xué)報(bào);2005年04期

6 劉毅;孫考祥;姚文軍;梁娜;慕宏杰;梁榮才;姚晨;;PAMAM包覆脂質(zhì)體的制備、表征及作為眼部遞藥載體的評(píng)價(jià)[J];高等學(xué)�；瘜W(xué)學(xué)報(bào);2010年04期

7 王勤;吳鐵;;PPI-DNA復(fù)合物的制備和PPI載體的細(xì)胞毒性研究[J];廣東藥學(xué)院學(xué)報(bào);2005年06期

8 蔣序林;;AQUEOUS SEC ANALYSIS OF CATIONIC POLYMERS AS GENE CARRIERS[J];Chinese Journal of Polymer Science;2011年04期

9 葉玲,顧微,周玉蘭;生物材料聚酰胺胺樹狀大分子在醫(yī)學(xué)領(lǐng)域研究進(jìn)展[J];高分子通報(bào);2002年04期

10 丁凡;邵增務(wù);;骨肉瘤基因治療載體研究進(jìn)展[J];國際骨科學(xué)雜志;2007年06期

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1 要e，

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