本文關(guān)鍵詞：精氨酸修飾的殼聚糖非病毒基因載體的研究　出處：《天津大學》2005年碩士論文　論文類型：學位論文

  更多相關(guān)文章： 殼聚糖 精氨酸修飾的殼聚糖 非病毒載體 基因轉(zhuǎn)染

【摘要】：殼聚糖是一種天然的陽離子多糖,具有良好的生物相容性,生物降解性,低免疫原性以及無毒性的優(yōu)點。殼聚糖通過靜電作用可以與DNA形成聚電解質(zhì)復合物,保護DNA免受核酸酶的降解,從而促使DNA順利的進入細胞,因此殼聚糖作為非病毒基因載體具有廣泛的應用前景。 本文制備了一種新型的精氨酸修飾的殼聚糖(Arg-CS)非病毒基因載體,研究內(nèi)容主要包括以下兩個方面: 1、精氨酸修飾的殼聚糖的制備及其與DNA復合物的表征。以碳二亞胺為偶聯(lián)劑,將精氨酸偶聯(lián)到殼聚糖上制備了精氨酸修飾的殼聚糖綴合物,并通過復凝聚的方法,制備了納米尺寸的Arg-CS/DNA聚電解質(zhì)復合物。通過紅外光譜和核磁共振圖譜分析,可以確認精氨酸已經(jīng)共價連接到殼聚糖上。圓二色譜結(jié)果表明DNA與殼聚糖作用形成納米尺寸的復合物后仍然保持著典型的B型構(gòu)象。用透射電鏡,原子力顯微鏡和光子相關(guān)光譜檢測復合物的表面形態(tài)和尺寸大小分布,結(jié)果表明,精氨酸修飾的殼聚糖/DNA納米復合物(N/P=2:1)呈現(xiàn)不規(guī)則的球形,復合物的直徑分布大多在80～150nm之間,有利于基因轉(zhuǎn)染。 2、細胞系和組織塊法培養(yǎng)血管平滑肌細胞,并以血管平滑肌細胞為宿主細胞,初步探討以精氨酸修飾的殼聚糖為載體,介導編碼綠色熒光蛋白質(zhì)粒轉(zhuǎn)染的可行性。倒置熒光顯微鏡下觀察到明亮清晰的綠色熒光,并且呈現(xiàn)出明顯的平滑肌細胞形態(tài),說明編碼綠色熒光蛋白的質(zhì)粒DNA被精氨酸修飾的殼聚糖載體成功地導入血管平滑肌細胞,并得到有效的表達。
[Abstract]:Chitosan is a kind of natural cationic polysaccharide , and has the advantages of good biocompatibility , biodegradability , low immunogenicity and no toxicity . Chitosan can form polyelectrolyte complex with DNA by electrostatic action , protect DNA from degradation of nucleases , thus promote the smooth entry of DNA into cells , so chitosan has wide application prospect as non - viral gene vector . A novel arginine - modified chitosan ( Arg - CS ) non - viral gene vector was prepared . 1 . Preparation of arginine - modified chitosan and characterization of the DNA complex . An arginine - modified chitosan conjugate was prepared by coupling arginine to chitosan by using carbodiimide as a coupling agent . The surface morphology and size distribution of the complex were determined by IR and NMR . The results showed that the chitosan / DNA nanocomposite ( N / P = 2 : 1 ) modified by arginine showed irregular spherical shape . The diameter distribution of the complex was mostly between 80 and 150 nm , which was beneficial to gene transfection . 2 銆，

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