本文關(guān)鍵詞：聚乙烯亞胺修飾納米金基因載體制備及體外實驗研究　出處：《第三軍醫(yī)大學(xué)學(xué)報》2016年09期 　論文類型：期刊論文

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【摘要】：目的制備聚乙烯亞胺修飾納米金基因載體并研究其理化性質(zhì)的表征參數(shù)和體外轉(zhuǎn)染效率。方法通過化學(xué)還原法制備聚乙烯亞胺修飾的納米金基因載體,用綠色熒光蛋白質(zhì)粒(pAc GFPN1)做報告基因,納米基因載體可通過靜電吸附的方式結(jié)合質(zhì)粒DNA。用紫外分光光度計檢測其吸收光譜,用透射電鏡觀察其形態(tài)特征,激光粒度分析儀測定其粒度分布、表面電位(Zeta電位),1%瓊脂糖凝膠電泳檢測該基因載體與質(zhì)粒DNA的結(jié)合穩(wěn)定性,CCK-8實驗檢測聚乙烯亞胺修飾納米金基因載體及DNA-納米金復(fù)合物對HEK293細(xì)胞的細(xì)胞毒性作用,通過熒光顯微鏡觀察聚乙烯亞胺納米基因載體介導(dǎo)pAcGFP-N1在體外培養(yǎng)的HEK293細(xì)胞中的表達(dá),并分析其轉(zhuǎn)染效率。結(jié)果聚乙烯亞胺還原氯金酸可以得到帶正電荷的納米顆粒,呈單分散球形分布,其粒徑為(12.3±3.3)nm。在pH=7.2時,Zeta電位為+(29.7±5.1)m V。1%瓊脂糖凝膠電泳結(jié)果表明,當(dāng)納米金/質(zhì)粒DNA≥0.5時,質(zhì)粒DNA可完全結(jié)合到納米金表面。體外轉(zhuǎn)染實驗表明,聚乙烯亞胺修飾納米金基因載體能介導(dǎo)pAcGFPN1轉(zhuǎn)染HEK293細(xì)胞并在細(xì)胞中表達(dá)綠色熒光蛋白,其轉(zhuǎn)染效率可達(dá)25%。結(jié)論聚乙烯亞胺修飾納米金是一種新型非病毒基因載體,具有轉(zhuǎn)染效率高、對細(xì)胞毒性小等優(yōu)勢。
[Abstract]:Objective to prepare polyvinyleneimine modified nano-gold gene vector and study its physical and chemical properties and transfection efficiency in vitro. Methods Polyvinyleneimine modified nano-gold gene vector was prepared by chemical reduction method. Using the green fluorescent protein particle pAc GFPN1 as the reporter gene, the nano-gene vector could bind to the plasmid DNA by electrostatic adsorption. The absorption spectrum of the vector was detected by UV spectrophotometer. The morphology of the plasmid was observed by transmission electron microscope, the particle size distribution was measured by laser particle size analyzer, and the binding stability of the gene vector to plasmid DNA was detected by 1% agarose gel electrophoresis. The cytotoxic effects of polyimide modified gold gene carrier and DNA-nano-gold complex on HEK293 cells were detected by CCK-8 assay. The expression of pAcGFP-N1 in HEK293 cells was observed by fluorescence microscope. The transfection efficiency was analyzed. Results the positively charged nanoparticles could be obtained by polyimide reduction with chloruronic acid, and the particle size was 12. 3 鹵3. 3nm. at pH=7.2. The Zeta potential was 29.7 鹵5.1mV.1% agarose gel electrophoresis. The results showed that the gold nanoparticles / plasmid DNA 鈮，

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