【作者】 郑钧丰；

【导师】 魏泓；

【作者基本信息】 第三军医大学 ， 动物学， 2008， 硕士

【摘要】 基因治疗是指将目的基因用基因转移技术导入目的细胞进行表达,使其获得特定的功能,从而达到治疗的目的[1]。基因治疗由三个方面组成:受体细胞、载体以及目的基因。皮肤成纤维细胞(dermal fibroblasts,DFs)获取方便,可以在体外大量扩增,容易进行遗传改造,并且具有容易移植和不易转化的优点,因此DFs成为目前基因治疗应用较为广泛的靶细胞,本论文亦将此细胞作为研究对象。目前,基因治疗面临的瓶颈问题在于转染效率低下,而决定基因治疗转染效率的关键点之一就在于载体的选择。目前基因治疗所应用的载体主要包括病毒载体和非病毒载体,其中应用最多的是病毒载体,包括逆转录病毒,腺病毒,腺相关病毒,疱疹病毒和慢病毒[2],其中前四种已经被美国FDA批准应用于临床。在人类基因治疗常用的病毒载体中,腺相关病毒( Adeno-associated viruses, AAV)的安全优势以及长效表达的优点[3-4]使其成为了基因稳定转染的宠儿。而腺病毒(Adenovirus, Ad)载体则因具有感染效率和外源基因表达水平高、高滴度制备较简单等特点而被广泛用于基因治疗研究和临床试验中[5-6]。因此,本论文选择AAV和Ad为研究对象。由于病毒载体的衣壳蛋白是决定其对靶细胞嗜性的主要因素,进行衣壳蛋白改造后的载体有望改变载体亲嗜性而提高转染效率。我们在选用常规使用的AAV2和Ad5的基础上,还选用了将AAV2的ITRs包被进入AAV1的衣壳蛋白形成的嵌合型载体——AAV2/1,以及将Ad35载体的纤突整合到Ad5载体上形成的Ad5/F35作为研究对象,通过比较他们的转染效率来筛选适合DFs的载体。本学位论文以eGFP为报告基因,以流式细胞检测(flow cytometry ,FCM)和荧光显微照相为研究手段,比较AAV2-eGFP、AAV2/1-eGFP、Ad5-eGFP和Ad5/F35-eGFP对DFs的转染效率,探讨了四种病毒载体转染DFs的适合条件,同时利用MTT法研究四种病毒载体转染后DFs的增殖性能,希望筛选出对DFs嗜性较高的载体,同时也进一步为成纤维细胞用于基因治疗的规模化生产应用提供实验和理论依据。AAV2-eGFP和AAV2/1-eGFP分别按照转染倍数MOI为104、105、106转染DFs,Ad5-eGFP和Ad5/F35-eGFP按照感染复数MOI为10、25、50、100转染DFs,转染后24h用流式细胞检测转染效率,荧光显微镜观察转染后的荧光强度,MTT检测两者对DFs增殖的影响。实验结果如下:AAV2/1-eGFP对DFs无感染能力,MOI 105时AAV2转染DFs 24h后转染效率可达到(22.16+5.59)%,表明AAV2转染DFs可以有效获得皮肤基因修饰细胞,进一步用于基因治疗和基因修饰。Ad5-eGFP和Ad5/F35-eGFP在MOI为10、25、50、100时转染效率随着MOI升高而呈上升趋势,MOI 100时,Ad5/F35-eGFP转染DFs24h后转染效率可达到90%左右,Ad5 -eGFP达到70%左右。综合实验结果分析,结论如下:Ad5/F35是四种载体中转染效率最高的载体,最适合实际生产应用和临床使用。AAV2载体能成功转染DFs,获得基因修饰细胞,本文所选的两种AAV载体不能解决转染效率低的问题。更多还原

【Abstract】 Gene therapy is the introduction of genetic material into cells for therapeutic purposes. There are three elements in gene therapy. They are vector, target cells and target gene.Dermal fibroblasts(DFs)are are convenient to obtain and prolific and hard to invert. These characteristics demonstrate that DFs are proper target cells which can be widely used in both gene therapy and gene modification.Recent findings suggest that major breakthroughs in low transfection rate for gene therapy are imminent, thus one of the key points is to use proper vector for gene delivery. The main vectors used in gene therapy include viral vectors and non-viral vectors. As viral vector, RV (retrovirus), Ad (Adenovirus), AAV (Adeno-associated virus) and HSV (herper simplex virus) have been used in clinic which is authorized by Food And Drug Administrtion (FDA) in USA. AAV vectors are prevalent for its stable transfection and can cause almost no pathogen reactions which make it prevalent in gene therapy especially for long-term expression. On the other hand, Adenoviral vectors can easily be obtained at high titers, thus Adenoviral vectors have been widely applied in gene therapy and gene functional study. On the basis of recent study, we investigate the transfection rate of AAV2 and AAV2/1 which inserted the ITRs of AAV2 on the AAV1 as well as Ad5 and Ad5/F35 which is integrated by the fiber nob of Ad35 and the Ad5 vector at different MOIs. The improved vectors may change tissue Addiction in gene delivery.In this investigation, DFs was isolated from human dermal skin which is authorized by related department and primarily cultured. AAV encoding enhanced green fluorescent protein (eGFP) were constructed and transfected in vitro to the human dermal fibroblasts (DFs) at MOI ranging from 1. 0×104 to 1.0×106v·g/ cell .Twenty four hours after the infection, the expression rates of eGFP on cultured DFs were assessed by flow cytometry and the transfected cells were observed by fluorescence microscope. Two AV encoding enhanced green fluorescent protein (eGFP) were constructed and transfected in vitro to the human dermal fibroblast at MOI 10, 25, 50 and 100.Twenty four hours after the infection, the expression rates of eGFP on cultured DFs were assessed by flow cytometry and the transfected cells were observed by fluorescence microscope. The killing effect of the four viruses on infective DFs was assayed respectively by MTT. The assays demonstrated following results:AAV2/1 has no efficiency when transfection DFs. The rate of AAV2 mediated transfection at MOI 105 referred to a peak of (22.16+5.59)% which demonstrated we can obtain gene transfer cells to apply in gene therapy and gene modification using AAV2.Transfection efficiency of Ad5-eGFP and Ad5/F35-eGFP were increased as MOI increased. At MOI 100, the rate of Ad5/F35 mediated transfection is about 90% whereas about 70% of Ad5The results demonstrate Ad5/F35 is the best vetor during the four vectors we investigated and we can obtain gene modification cells for gene therapy using AAV2 with low transfectiong efficiency which means the low efficiency remain a problem on AAV.更多还原