本文選題：葉酸 + 殼聚糖　； 參考：《武漢工業(yè)學院》2009年碩士論文

【摘要】： 目前,殼聚糖作為一種新型非病毒基因載體,已引起越來越多的國家和研究機構的重視。葉酸—葉酸受體靶向是近年來倍受青睞的一種新型抗腫瘤機制,它是利用葉酸受體在某些腫瘤部位的過度表達而在正常組織低水平表達的特性而實現(xiàn)目的基因的靶向輸送。因此,用葉酸對殼聚糖進行化學修飾,可提高基因轉染的靶向性和轉染效率。 本實驗利用殼聚糖氮原子上的�；磻�,縮合劑首先與葉酸生成活性中間體,進一步與殼聚糖上的氨基反應制備葉酸偶聯(lián)殼聚糖。實驗中制備了不同連接比例的葉酸偶聯(lián)殼聚糖,采用紅外光譜法驗證葉酸與殼聚糖的偶聯(lián),用紫外分光光度法測定每個殼聚糖上連接葉酸的數(shù)量;選擇綠色熒光蛋白基因作為報告基因,用復凝聚法制備葉酸偶聯(lián)殼聚糖-DNA納米粒和殼聚糖-DNA納米粒,用透射電鏡觀察納米微粒表面形態(tài),通過DNA酶保護性檢測實驗觀察葉酸偶聯(lián)殼聚糖-DNA納米粒和殼聚糖-DNA納米粒對質粒的保護作用;利用培養(yǎng)的HeLa細胞進行轉染,通過熒光顯微鏡定性觀察,比較葉酸偶聯(lián)殼聚糖納米粒和殼聚糖納米粒作為基因治療載體的優(yōu)劣。 葉酸偶聯(lián)殼聚糖紅外光譜圖表明葉酸與殼聚糖成功偶聯(lián),每個殼聚糖上連接葉酸的數(shù)量大約為3個、10個、20個、30個;實驗中制備的葉酸偶聯(lián)殼聚糖-DNA納米粒溶液和殼聚糖-DNA納米粒溶液為外觀均一、穩(wěn)定的透明溶液;透射電子顯微鏡檢測結果顯示,納米粒分布均勻,大小一致,無粘合,外觀呈球形或近球形,葉酸偶聯(lián)殼聚糖-DNA納米粒粒徑大約為120nm左右,殼聚糖-DNA納米粒粒徑大約為140nm左右;DNA酶保護性檢測實驗表明殼聚糖納米粒和葉酸偶聯(lián)殼聚糖納米粒均有良好的DNA保護作用;熒光顯微鏡檢測結果顯示,葉酸偶聯(lián)殼聚糖-DNA納米粒和殼聚糖-DNA納米粒均能將綠色熒光蛋白質�；蜻f送到細胞內并表達產生綠色熒光蛋白,但每個殼聚糖上連接10個和20個葉酸的葉酸偶聯(lián)殼聚糖-DNA納米粒轉染效果更好,有更高的靶向性和轉染效率。
[Abstract]:At present, chitosan as a novel non viral gene vector, has attracted more and more countries and research institutions pay much attention. Folic acid - folate receptor targeting is a new anti-tumor mechanism in recent years is popular, it is characteristic in the over expression of some kinds of tumor and normal tissue expression in the low level of use of folate receptor the realization of the target of gene transfer. Therefore, the chemical modification of chitosan with folic acid, can improve the targeting of gene transfection and transfection efficiency.
This experiment using the acylation reaction of Chitosan on nitrogen atom, condensation agent and folic acid first generation of reactive intermediates, further reacted with amino groups of the preparation of Chitosan on Preparation of folate conjugated chitosan. Weprepared different folate conjugated chitosan connection ratio, coupling by using infrared spectroscopy to prove the amount of folate with chitosan. The connection of folic acid in each chitosan was determined by UV spectrophotometry; selection of green fluorescent protein gene as a reporter gene, using complex coacervation of folate conjugated chitosan -DNA nanoparticles and chitosan -DNA nanoparticles by using transmission electron microscope observation of surface morphology of nano particles, the DNA enzyme protection assay. The protective effect of folate chitosan -DNA nanoparticles and chitosan -DNA nanoparticles on plasmid; transfection by cultured HeLa cells by fluorescence microscope, comparison of folate conjugated chitosan Nanoparticles and chitosan nanoparticles are used as gene therapy carriers.
Folate conjugated chitosan IR spectra indicate that the coupling of folate with chitosan was successful, chitosan on the number of connections each folic acid is about 3, 10, 20, 30; in the experiment of preparation of folate conjugated chitosan -DNA nanoparticles solution and chitosan -DNA nanoparticles solution for appearance are a stable, transparent solution; transmission electron microscopy showed that nanoparticles of uniform distribution, the same size, no adhesion, the appearance of a spherical or nearly spherical, folate coupled chitosan -DNA nanoparticle size is about 120nm, chitosan -DNA nanoparticle size is about 140nm; DNA enzyme detection experiments show that DNA protective protective effect of chitosan nanoparticles and folic acid conjugated chitosan nanoparticles were good; fluorescence microscopy showed that folate conjugated chitosan -DNA nanoparticles and chitosan -DNA nanoparticles can be green fluorescent protein gene delivery The green fluorescent protein was expressed in the cells, but the folate conjugated chitosan -DNA nanoparticles with 10 folate and 20 folate on each chitosan had better transfection efficiency and higher targeting and transfection efficiency.

【學位授予單位】：武漢工業(yè)學院
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R346

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