DNA分子的链式扩增、组装及载药研究

发布时间：2018-04-06 07:45

本文选题：DNA　切入点：扩增　出处：《西南大学》2014年硕士论文

【摘要】：一直以来,DNA都是分子生物学、生物化学、药物分析等学科研究领域的明星分子。人们试图通过努力多方位地认识DNA分子,以便更好地利用DNA分子来改造客观世界。因此,精确地分析DNA并挖掘其潜在的生物学功能很有研究价值。本文试图建立一种简单灵敏的短链DNA扩增方法,并进一步将DNA分子优异的生物学性能用于材料组装和靶向药物运载中。具体研究内容如下： (1)利用连接酶介导的聚合酶链式反应(PCR)技术建立短链DNA的分析方法。PCR常用于临床检测病毒、细菌、细胞因子等的DNA或者RNA,但是通常只用于分析长链DNA序列,不能用于短链DNA的直接检测。我们通过实验设计了各自与靶物DNA一半互补的长度均为52个碱基的长链DNA作为检测探针,在T4DNA连接酶的作用下,这两段探针能以短链靶物DNA为模板连成104个碱基的长链,再将长链用PCR扩增,就能实现对短链靶物DNA的间接放大检测。借助DNA的特异性杂交作用以及PCR的扩增作用,这段只有16个碱基的短链DNA的检测限低至100fM,线性范围跨越5个数量级,而且这种方法的抗干扰能力强,即使在复杂生物介质细胞裂解液中也能灵敏地分析靶物DNA。 (2)利用杂交链式反应(HCR)生成的长链DNA组装金纳米颗粒(AuNPs).纳米材料组装体因其微米尺寸而呈现出一些优于单体的光电性质,成为了目前研究的热点。杂交链式反应是一种新的DNA自组装技术,它提供了一种合成长链DNA的简单方法。基于杂交链式反应生成的长链DNA,本文建立了一种简单的纳米材料的组装方法,只需通过金纳米颗粒与巯基DNA之间形成共价键,就能实现金纳米颗粒沿长链DNA的组装,其中长链DNA是通过杂交链式反应生成的。通过控制杂交链式反应的时间,可以得到长短不一的DNA产物,再与金纳米颗粒作用后,生成各种长度的组装体。研究发现,这种链状的金纳米颗粒组装体能清晰地在暗场显微镜下成像,而单个金纳米颗粒的散射信号非常微弱。这表明,组装后材料的光散射性质大大提高。 (3)金纳米颗粒介导的DNA负载阿霉素(Dox)用于癌症的靶向治疗。临床癌症治疗中存在的主要问题有：药物对正常组织的副作用大,药物有效利用度低且耐受性差。因此建立有效的靶向给药系统,克服以上不足,对癌症的治疗很有意义。叶酸是一种普遍研究的靶向配体,而对于一些不表达或者少量表达叶酸受体的癌细胞,寻找一些其他的靶向配体用于提高药物的疗效有必要。我们通过在金纳米颗粒表面修饰上含有朊蛋白核酸适配子的DNA用于负载阿霉素,建立一种靶向治疗母细胞瘤的给药系统。借助于核酸适配子与神经瘤母细胞的特异性识别作用,导致DNA构型发生改变,在癌症部位释放出阿霉素,特异性地杀死癌细胞。细胞毒性实验和细胞荧光成像实验很好地验证了这种针对母细胞瘤的给药系统的有效性。综上所述,本文首先建立了一种简单快速的链式扩增短链DNA的方法,然后再利用DNA特殊的生物学性质结合金纳米颗粒优良生物相容性及光电性质,将它们应用于纳米材料组装及靶向药物运载领域。本研究的优势在于：一是利用了PCR技术间接实现对短链DNA的灵敏分析：二是利用杂交链式发应生成带巯基的长链DNA,通过Au-S键的作用,将金纳米颗粒组装到DNA上；三是利用核酸适配子作为靶向配体,将阿霉素运送到神经瘤母细胞中实现靶向治疗癌症。本研究为建立新的DNA分析方法及探索其进一步应用提供了参考价值。
[Abstract]:DNA has been a star in the fields of molecular biology , biochemistry , drug analysis , etc . It has been tried to make better use of DNA molecules to transform the objective world . Therefore , it is valuable to accurately analyze DNA and to explore its potential biological functions . This paper attempts to establish a simple and sensitive short - strand DNA amplification method and to further improve the biological properties of DNA molecules for material assembly and targeting drug delivery . The specific research contents are as follows :

( 1 ) Using ligase - mediated polymerase chain reaction ( PCR ) technique to establish a short - chain DNA analysis method . PCR is used to detect DNA or RNA of virus , bacteria , cytokines , etc . It is usually used to analyze long - chain DNA sequence and can not be used for the direct detection of short - strand DNA . By means of specific hybridization of DNA and PCR amplification , the detection limit of short - strand DNA of 16 bases is low to 100fM , and the linear range is over 5 orders of magnitude , and the method has strong anti - interference ability , and can be used to analyze target DNA sensitively even in complex biological medium cell lysate .

This paper presents a simple method for the synthesis of long - chain DNA by means of hybrid chain reaction .

( 3 ) Targeting therapy for cancer by gold nanoparticles mediated by adriamycin ( Dox ) . The main problems in the treatment of cancer are that the drug has a large side effect on normal tissues , low effective bioavailability and poor tolerance . It is necessary to establish an effective targeting ligand for cancer .

In conclusion , this paper first establishes a simple and rapid method to amplify short - strand DNA , then uses the special biological properties of DNA to combine the excellent biocompatibility and photoelectric properties of gold nanoparticles , and applies them to the field of nano - material assembly and targeting drug delivery . The research has the advantage that : firstly , the long - chain DNA with sulfydryl should be generated by means of PCR technique , and gold nanoparticles are assembled onto DNA by the action of Au - S bond ;
Third , using the nucleic acid aptamer as the targeting ligand , the adriamycin is transported to the neuroma mother cell to achieve the targeted treatment of cancer . The research provides a reference value for establishing a new DNA analysis method and exploring the further application thereof .

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R917

【共引文献】