本文選題：葉酸 + 殼聚糖��； 參考：《武漢工業(yè)學(xué)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(lái)越多的國(guó)家和研究機(jī)構(gòu)的重視。葉酸—葉酸受體靶向是近年來(lái)倍受青睞的一種新型抗腫瘤機(jī)制,它是利用葉酸受體在某些腫瘤部位的過(guò)度表達(dá)而在正常組織低水平表達(dá)的特性而實(shí)現(xiàn)目的基因的靶向輸送。因此,用葉酸對(duì)殼聚糖進(jìn)行化學(xué)修飾,可提高基因轉(zhuǎn)染的靶向性和轉(zhuǎn)染效率。 本實(shí)驗(yàn)利用殼聚糖氮原子上的�；磻�(yīng),縮合劑首先與葉酸生成活性中間體,進(jìn)一步與殼聚糖上的氨基反應(yīng)制備葉酸偶聯(lián)殼聚糖。實(shí)驗(yàn)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(yàn)證葉酸與殼聚糖的偶聯(lián),用紫外分光光度法測(cè)定每個(gè)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(bào)告基因,用復(fù)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(guò)DNA酶保護(hù)性檢測(cè)實(shí)驗(yàn)觀察葉酸偶聯(lián)殼聚糖-DNA納米粒和殼聚糖-DNA納米粒對(duì)質(zhì)粒的保護(hù)作用;利用培養(yǎng)的HeLa細(xì)胞進(jìn)行轉(zhuǎn)染,通過(guò)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(gè)殼聚糖上連接葉酸的數(shù)量大約為3個(gè)、10個(gè)、20個(gè)、30個(gè);實(shí)驗(yàn)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(cè)結(jié)果顯示,納米粒分布均勻,大小一致,無(wú)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(hù)性檢測(cè)實(shí)驗(yàn)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(hù)作用;熒光顯微鏡檢測(cè)結(jié)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(zhì)粒基因遞送到細(xì)胞內(nèi)并表達(dá)產(chǎn)生綠色熒光蛋白,但每個(gè)殼聚糖上連接10個(gè)和20個(gè)葉酸的葉酸偶聯(lián)殼聚糖-DNA納米粒轉(zhuǎn)染效果更好,有更高的靶向性和轉(zhuǎn)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(xué)位授予單位】：武漢工業(yè)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R346

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