【摘要】：近年來,由于基因治療在替代功能障礙基因和腫瘤治療方面的潛在應(yīng)用前景而備受關(guān)注�；蛑委煹年P(guān)鍵問題之一在于開發(fā)安全、高效的基因轉(zhuǎn)染體系。在轉(zhuǎn)基因載體的研究發(fā)展過程中,一個明顯的趨勢是采用非病毒轉(zhuǎn)基因載體,其目的在于降低人體正常細(xì)胞致病的風(fēng)險。 本文選用天然無毒、可生物降解、具有良好生物相容性的帶正電荷高分子材料——殼聚糖作為載體,通過納米生物技術(shù)制備了載質(zhì)粒的殼聚糖-質(zhì)粒納米粒。系統(tǒng)地研究了殼聚糖分子量,N/P,pH值,制備方法等因素對殼聚糖-pDNA納米粒的形態(tài)、包埋率、載藥率、基因藥物在摸擬體內(nèi)環(huán)境下的釋放的影響。以優(yōu)化殼聚糖-pDNA納米粒的制備條件,探討制備條件與殼聚糖-pDNA納米粒的性質(zhì)相關(guān)性,進而揭示殼聚糖-pDNA納米粒作為基因治療的載體的機理所在。 殼聚糖的結(jié)晶性、力學(xué)特性、通透性等都和殼聚糖分子量大小相關(guān),而殼聚糖的許多獨特功能只有在分子量降低到一定程度時才表現(xiàn)出來。因此,選擇適當(dāng)?shù)姆椒▽ぞ厶沁M行降解就顯得尤為重要。本文選用超聲波技術(shù)與H_2O_2氧化降解法相結(jié)合的方法對殼聚糖進行降解以獲得純化的不同分子量的殼聚糖。結(jié)果表明:在30℃,采用降解體系為4%的殼聚糖的5%乙酸溶液,加入H_2O_2與糖單元摩爾比為1.0,進行超聲波輔助降解40min-60min可獲得滿意的結(jié)果。 殼聚糖分子在酸性條件下荷正電,DNA分子在堿性條件下荷負(fù)電,我們利用這一特點采用復(fù)凝聚法制備基因殼聚糖納米粒。以納米粒的平均粒徑與形態(tài)作為控制指標(biāo),進行單因素實驗。從納米粒的粒徑分布、形態(tài)、包埋率、載藥率、基因包埋量和體外釋放率等方面考察了納米粒的基本性質(zhì)。結(jié)果表明:采用復(fù)凝聚制備法能夠?qū)崿F(xiàn)殼聚糖對質(zhì)粒的包埋,包埋率達到83.46%。制備的殼聚糖質(zhì)粒納米粒呈亞球型,粒徑在300～600nm。殼聚糖質(zhì)粒納米粒
[Abstract]:In recent years, gene therapy has attracted much attention because of its potential application in gene substitution for dysfunction and tumor therapy. One of the key problems of gene therapy is to develop a safe and efficient gene transfection system. In the process of research and development of transgenic vectors, there is an obvious trend to adopt non-viral transgenic vectors, which aims to reduce the risk of human normal cell pathogenicity. In this paper, chitosan, a naturally nontoxic, biodegradable and biocompatible positively charged polymer material, was used as carrier to prepare plasmide-loaded chitosan plasmid nanoparticles. The effects of molecular weight of chitosan, pH value of N / P, and preparation method on the morphology, entrapment rate, drug loading rate and drug delivery rate of chitosan pDNA nanoparticles were systematically studied. In order to optimize the preparation conditions of chitosan-pDNA nanoparticles, the relationship between the preparation conditions and the properties of chitosan-pDNA nanoparticles was discussed, and the mechanism of chitosan-pDNA nanoparticles as the carrier of gene therapy was revealed. The crystallinity, mechanical properties and permeability of chitosan are all related to the molecular weight of chitosan. However, many unique functions of chitosan are only shown when the molecular weight decreases to a certain extent. Therefore, it is very important to choose suitable method to degrade chitosan. In this paper, ultrasonic technology combined with H_2O_2 oxidation degradation method was used to degrade chitosan to obtain purified chitosan with different molecular weight. The results showed that at 30 鈩，

本文編號：2139713