壳聚糖增强重组腺病毒转导效率及口服免疫效果初步研究

发布时间：2018-03-30 10:47

  本文选题：重组腺病毒　切入点：壳聚糖　出处：《北京协和医学院》2009年博士论文

【摘要】：5型重组腺病毒载体(rAdv)因具有基因转导效率高、病毒载体滴度高及能感染非复制相细胞等多种优点而得到广泛应用,目前我国已经有两种基于腺病毒的抗肿瘤药物得到批准。但由于rAdv的基因转导主要是通过柯萨奇病毒-腺病毒受体(CAR)介导,导致rAdv对CAR受体表达缺失的细胞基因转导效率低下。因此研究非CAR受体依赖的、高效、低毒及简单易行的促进rAdv基因转导效率的方法十分必要。 壳聚糖是一种天然的阳离子多糖,具有无毒、生物相容性好、生物可降解及无免疫原性等多种优点。但是其在生理pH条件下(pH7.4)不溶的特性限制了其生物学应用。已有的壳聚糖促进rAdv基因转导效率的实验需要特殊的酸性pH条件(pH6.4),限制了其应用,需要开发新的方法。鉴此,本研究拟探索在生理pH条件下促进rAdv的基因转导效率新的壳聚糖溶解方法,阐明其促进基因转导能力,并在小鼠体内测试其促进口服rAdv免疫效果,为壳聚糖用于促进rAdv介导的基因治疗和疫苗免疫效果提供基础。 本研究首先建立了促进壳聚糖在pH7.4条件下溶解的方法。经过大量筛选,我们发现利用NaHCO3溶液可以实现壳聚糖在生理pH条件下的溶解。结果显示,该壳聚糖溶液具有明显的生物学效应,可以在生理pH条件下显著促进rAdv在多种细胞上的基因转导效率。在CAR受体缺陷的细胞(CHO、DC2.4、RD、B16细胞系)中,壳聚糖碳酸氢钠溶液可以将rAdv的基因转导效率提高8-20倍；而在CAR受体高表达的细胞系(A549和HepⅡ),壳聚糖碳酸氢钠溶液也可以将rAdv的基因转导效率提高1.5倍左右。壳聚糖碳酸氢钠溶液的促进转导作用不受培养基中血清及rAdv纯度的影响,对抗Ad5血清也有一定的耐受性。说明壳聚糖碳酸氢钠溶液可以非CAR受体依赖、高效、低毒及简便易行的促进rAdv基因转导效率。 在观察了壳聚糖碳酸氢钠溶液在体外促进rAdv基因转导效率之后,我们观察该新溶液是否具有在体应用的价值。我们发现在壳聚糖碳酸氢钠溶液基础上研制的壳聚糖制剂可以显著提高rAdv对模拟胃液环境的耐受性,且该作用可以被海藻糖进一步加强。相对于口服单独给予表达轮状病毒VP6基因的重组腺病毒(rAdv-VP6)疫苗,口服给予小鼠rAdv-VP6疫苗的壳聚糖制剂可以显著提高免疫后小鼠血清抗体阳转率以及抗体滴度,并可诱导粘膜器官抗原特异性IgA反应。轮状病毒攻击保护试验证实其能显著降低轮状病毒攻击后小鼠粪便中的排毒量。结果表明,该壳聚糖制剂可以显著提高口服rAdv-VP6的免疫效果及攻毒保护效果。 综上所述,本研究首次建立了一种能促进壳聚糖在生理pH条件下溶解的方法,该溶液不仅在体外可以促进rAdv的基因转导效率,其相应口服制剂还可以增强口服重组腺病毒的免疫效果及保护作用,为其在基因治疗和重组疫苗中的应用提供了基础。
[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，

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