本文選題：重組腺病毒　切入點：殼聚糖　出處：《北京協(xié)和醫(yī)學(xué)院》2009年博士論文

【摘要】：5型重組腺病毒載體(rAdv)因具有基因轉(zhuǎn)導(dǎo)效率高、病毒載體滴度高及能感染非復(fù)制相細胞等多種優(yōu)點而得到廣泛應(yīng)用,目前我國已經(jīng)有兩種基于腺病毒的抗腫瘤藥物得到批準(zhǔn)。但由于rAdv的基因轉(zhuǎn)導(dǎo)主要是通過柯薩奇病毒-腺病毒受體(CAR)介導(dǎo),導(dǎo)致rAdv對CAR受體表達缺失的細胞基因轉(zhuǎn)導(dǎo)效率低下。因此研究非CAR受體依賴的、高效、低毒及簡單易行的促進rAdv基因轉(zhuǎn)導(dǎo)效率的方法十分必要。 殼聚糖是一種天然的陽離子多糖,具有無毒、生物相容性好、生物可降解及無免疫原性等多種優(yōu)點。但是其在生理pH條件下(pH7.4)不溶的特性限制了其生物學(xué)應(yīng)用。已有的殼聚糖促進rAdv基因轉(zhuǎn)導(dǎo)效率的實驗需要特殊的酸性pH條件(pH6.4),限制了其應(yīng)用,需要開發(fā)新的方法。鑒此,本研究擬探索在生理pH條件下促進rAdv的基因轉(zhuǎn)導(dǎo)效率新的殼聚糖溶解方法,闡明其促進基因轉(zhuǎn)導(dǎo)能力,并在小鼠體內(nèi)測試其促進口服rAdv免疫效果,為殼聚糖用于促進rAdv介導(dǎo)的基因治療和疫苗免疫效果提供基礎(chǔ)。 本研究首先建立了促進殼聚糖在pH7.4條件下溶解的方法。經(jīng)過大量篩選,我們發(fā)現(xiàn)利用NaHCO3溶液可以實現(xiàn)殼聚糖在生理pH條件下的溶解。結(jié)果顯示,該殼聚糖溶液具有明顯的生物學(xué)效應(yīng),可以在生理pH條件下顯著促進rAdv在多種細胞上的基因轉(zhuǎn)導(dǎo)效率。在CAR受體缺陷的細胞(CHO、DC2.4、RD、B16細胞系)中,殼聚糖碳酸氫鈉溶液可以將rAdv的基因轉(zhuǎn)導(dǎo)效率提高8-20倍；而在CAR受體高表達的細胞系(A549和HepⅡ),殼聚糖碳酸氫鈉溶液也可以將rAdv的基因轉(zhuǎn)導(dǎo)效率提高1.5倍左右。殼聚糖碳酸氫鈉溶液的促進轉(zhuǎn)導(dǎo)作用不受培養(yǎng)基中血清及rAdv純度的影響,對抗Ad5血清也有一定的耐受性。說明殼聚糖碳酸氫鈉溶液可以非CAR受體依賴、高效、低毒及簡便易行的促進rAdv基因轉(zhuǎn)導(dǎo)效率。 在觀察了殼聚糖碳酸氫鈉溶液在體外促進rAdv基因轉(zhuǎn)導(dǎo)效率之后,我們觀察該新溶液是否具有在體應(yīng)用的價值。我們發(fā)現(xiàn)在殼聚糖碳酸氫鈉溶液基礎(chǔ)上研制的殼聚糖制劑可以顯著提高rAdv對模擬胃液環(huán)境的耐受性,且該作用可以被海藻糖進一步加強。相對于口服單獨給予表達輪狀病毒VP6基因的重組腺病毒(rAdv-VP6)疫苗,口服給予小鼠rAdv-VP6疫苗的殼聚糖制劑可以顯著提高免疫后小鼠血清抗體陽轉(zhuǎn)率以及抗體滴度,并可誘導(dǎo)粘膜器官抗原特異性IgA反應(yīng)。輪狀病毒攻擊保護試驗證實其能顯著降低輪狀病毒攻擊后小鼠糞便中的排毒量。結(jié)果表明,該殼聚糖制劑可以顯著提高口服rAdv-VP6的免疫效果及攻毒保護效果。 綜上所述,本研究首次建立了一種能促進殼聚糖在生理pH條件下溶解的方法,該溶液不僅在體外可以促進rAdv的基因轉(zhuǎn)導(dǎo)效率,其相應(yīng)口服制劑還可以增強口服重組腺病毒的免疫效果及保護作用,為其在基因治療和重組疫苗中的應(yīng)用提供了基礎(chǔ)。
[Abstract]:Recombinant adenovirus vector type 5 is widely used because of its high efficiency of gene transduction, high titer of virus vector and ability to infect non-replicating phase cells. At present, two kinds of adenovirus-based antitumor drugs have been approved in China. However, the gene transduction of rAdv is mainly mediated by coxsackievirus (Coxsackievirus)-adenovirus receptor (car). Therefore, it is necessary to study non-#en2# receptor dependent, efficient, low toxicity and simple methods to promote the efficiency of rAdv gene transduction. Chitosan is a kind of natural cationic polysaccharide, which is nontoxic and has good biocompatibility. Biodegradable and non-immunogenicity, however, its insoluble properties at physiological pH (pH7.4) limit its biological application. The experiments of chitosan promoting rAdv gene transduction efficiency need special acidic pH strip. Its application is limited by its pH 6.4. Therefore, we aim to explore a new chitosan dissolution method to promote the gene transduction efficiency of rAdv at physiological pH, to elucidate its ability to promote gene transduction, and to test its effect on oral rAdv immunization in mice. It provides the basis for the application of chitosan in promoting rAdv mediated gene therapy and vaccine immunization. In this study, we first established a method to promote the dissolution of chitosan under pH7.4 conditions. After a lot of screening, we found that using NaHCO3 solution can realize the dissolution of chitosan under physiological pH conditions. The chitosan solution has obvious biological effect and can significantly promote the gene transduction efficiency of rAdv on many kinds of cells under physiological pH condition. Chitosan sodium bicarbonate solution could increase the gene transduction efficiency of rAdv by 8-20 times. In CAR receptor overexpression cell lines such as A549 and Hep 鈪，

本文編號：1685587