多功能核酸纳米载药系统对肿瘤的疗效检测
发布时间：2018-08-25 19:58
【摘要】：癌症的诊断和治疗在生化分析领域研究中备受关注,涉及的方法也越来越具有丰富性和创新性,其中关于功能核酸纳米体系已呈现出较好的研究和应用前景。本文设计了一种多功能核酸纳米体系,一方面,利用DNA自组装原理,通过滚环扩增(Rolling Circle Amplification,RCA)技术和末端转移反应,形成了多功能DNA纳米球体系,作为抗癌药物阿霉素(DOX)的载体,用于对不同种癌细胞的特异性识别和靶向给药;同时还可用于细胞中还原性巯基化合物的含量测定。实验结果表明本DNA纳米球材料具有良好的生物相容性和较强的靶向选择性,降低正常细胞损伤的同时实现了对癌细胞的杀灭作用,对谷胱甘肽等还原性巯基化合物的检测具有灵敏度高、重复性好的良好特性;另一方面,本文还建立在滚环扩增方法的基础之上,利用邻近连接效应(proximity ligation,PLA),进一步与氧化石墨烯和介孔二氧化硅这两种无机纳米材料相结合,实现了细胞内小分子物质的检测和胞内对药物的释放这两个过程的进行。该设计有效降低了生物体内除检测样本外无关物质的消化作用影响,使检测更具可行性和高灵敏性。其主要内容如下:1.基于DNA自组装技术和末端转移反应的多功能磁性DNA纳米球对谷胱甘肽的检测和抗癌药物DOX的靶向输送:首先,利用DNA自组装技术原理,通过滚环扩增方法,形成DNA纳米球。其次,在合成DNA纳米球的基础上,引入磁性微球,形成磁性DNA纳米球,便于分离。通过对参与组装的DNA引物链进行改造,可使其与含有双硫键的DNA单链进行杂交反应,从而形成了含有双硫键的DNA纳米球,该纳米球可实现对细胞内谷胱甘肽等还原性巯基化合物含量的检测,检测灵敏度高。再者,通过对合成DNA纳米球过程中参与滚环扩增过程的模板链进行改造,使其含有sgc8核酸适配体序列,该序列可特异性识别CEM细胞上过度表达的PTK7蛋白,由此合成的含sgc8核酸适配体序列的DNA纳米球可实现对CEM细胞的选择性药物输送。最后,建立在DNA纳米球的基础上,我们还通过末端转移酶的作用,在滚环扩增形成的线性长链末端转移上多个腺嘌呤(A)碱基,此末端可与修饰有叶酸的多聚胸腺嘧啶(T)单链进行杂交反应,从而形成了含有叶酸的DNA纳米球,该纳米球可对那些会在表面过度表达叶酸受体的癌细胞,如HeLa细胞等进行特异性识别,实现药物的有效输送。2.基于滚环扩增方法和邻近连接效应的信号放大检测ATP并诱导介孔二氧化硅开孔实现药物释放的体系:邻近连接(proximity ligation,PLA)可将两条不同的单链DNA分子分别与同一个蛋白质识别分子相结合,使得两条DNA单链的尾部可在空间上紧密靠近,在DNA连接酶的作用下,游离的5’端和3’端发生连接反应,从而形成滚环扩增方法所必需的环状结构。因此我们设计了一条含有ATP适体序列的DNA单链作为连接链,一条可与连接链进行末端互补的模板链,一条可与模板链的序列部分相似的信号链。三种DNA单链同时吸附到氧化石墨烯纳米材料上,具有良好生物相容性和纳米级粒径的氧化石墨烯可通过胞吞作用进入癌细胞。该体系一旦与ATP分子相遇,即可发生ATP适体链部分与ATP分子的结合作用,从而发生邻近连接效应,并在DNA连接酶的作用下形成环状DNA复合物,进而完成滚环扩增过程,所形成的长线性DNA单链含有成千上万的碱基,可结合大量附着在氧化石墨烯上的信号链,释放出较强的荧光。通过对释放的荧光进行检测来实现对ATP浓度的测量,检测信号强、灵敏度高。同时,为了提高该系统的选择性,我们在氧化石墨烯的表面修饰了叶酸分子,可与过度表达叶酸受体的癌细胞,如HeLa细胞进行特异性结合,在与缺乏叶酸受体的MCF-7细胞的对比实验中,表现出明显的较高选择性特点,实验结果进一步说明了本系统在癌症诊断中的应用潜能;另一方面,我们又设计了一条与上述系统中所提及的信号链碱基序列一致但不含荧光基团的DNA单链,将该DNA单链通过静电吸引作用吸附到介孔硅的微孔道口,作为门DNA单链,封锁微孔道口,封装介孔二氧化硅内部预先装备好的药物DOX。该系统所形成封闭的药物运载及控释结构,只有在ATP的作用下,发生上述滚环扩增过程后所形成的线性长链出现时,可能与门DNA单链杂交,使其脱离介孔二氧化硅表面,将微孔上的“门”打开,从而释放出药物。这一设计可实现对药物的可控释放,有效降低阿霉素对正常细胞的强毒副作用,具有很强的实用性。同样地,为了提高该系统的特异性选择能力,对介孔二氧化硅表面进行叶酸的修饰,实现对过度表达叶酸受体的癌细胞的特异性识别,进而靶向给药。该体系有效降低了目标分子外其他物质的消化影响,并降低了抗癌药物的毒副作用,使得检测方法更具高灵敏性、选择性和可行性。
[Abstract]:Cancer diagnosis and treatment have attracted much attention in the field of biochemical analysis, and the methods involved are becoming more and more rich and innovative. Among them, functional nucleic acid nanosystems have shown good research and application prospects. Rolling Circle Amplification (RCA) and terminal metastasis reactions have led to the formation of a multifunctional DNA nanosphere system as a carrier of doxorubicin (DOX) for the specific recognition and targeted drug delivery of various cancer cells, and also for the determination of reducing sulfhydryl compounds in cells. Nanospheres have good biocompatibility and strong targeting selectivity, which can reduce the damage of normal cells and kill cancer cells at the same time. They have high sensitivity and good reproducibility for the detection of glutathione and other reductive sulfhydryl compounds. On the other hand, this paper is based on the roller amplification method. Furthermore, proximity ligation (PLA) was used to combine with graphene oxide and mesoporous silica to realize the detection of intracellular small molecules and the release of intracellular drugs. The main contents are as follows: 1. Detection of glutathione and targeted delivery of antitumor drug DOX by multifunctional magnetic DNA nanospheres based on DNA self-assembly technology and end-transfer reaction: Firstly, using the principle of DNA self-assembly technology, DNA nanoparticles were formed by rolling amplification method. Secondly, magnetic DNA nanospheres were introduced to form magnetic DNA nanospheres on the basis of synthesizing DNA nanospheres for easy isolation. DNA primer chains were modified to hybridize DNA single strands containing disulfide bonds, thus forming DNA nanospheres containing disulfide bonds. The nanospheres were able to achieve intracellular glutathione. Furthermore, the template chain involved in the roll-ring amplification process during the synthesis of DNA nanospheres was modified to contain the sgc8 aptamer sequence, which can specifically identify the over-expressed PTK7 protein on CEM cells and thus synthesize the aptamer containing sgc8 nucleic acid. Finally, on the basis of DNA nanospheres, we also transfer a number of adenine (A) bases to the linear long chain end formed by ring-rolling amplification, which can be hybridized with the polythymine (T) single strand modified with folic acid, by the action of terminal transferase. DNA nanospheres containing folic acid were formed by the reaction. These nanospheres can identify the cancer cells which overexpress folic acid receptors on the surface, such as HeLa cells, to achieve effective drug delivery. Drug release system: Proximity ligation (PLA) can bind two different single-stranded DNA molecules to the same protein recognition molecule, so that the tail of the two single strands of DNA can be closely spaced. Under the action of DNA ligase, the free 5'and 3'ends of the DNA chain react to form a ring-rolling amplification. Therefore, we have designed a DNA single strand containing ATP aptamer sequence as a link chain, a template chain which can complement the end of the link chain, and a signal chain which can partly resemble the sequence of the template chain. Once the system meets the ATP molecule, it can bind the aptamer chain of ATP to the ATP molecule, which results in the adjacent junction effect, and forms a circular DNA complex under the action of DNA ligase, thus completing the rolling amplification process. Long linear DNA single strands contain thousands of bases, which can bind to a large number of signal chains attached to graphene oxide and emit strong fluorescence. The ATP concentration can be measured by detecting the fluorescence. The detection signal is strong and the sensitivity is high. At the same time, in order to improve the selectivity of the system, we repair the surface of graphene oxide. Folic acid molecule can specifically bind to cancer cells with over-expression of folate receptor, such as HeLa cells. Compared with MCF-7 cells lacking folate receptor, it shows obvious high selectivity. The experimental results further illustrate the potential of this system in cancer diagnosis. On the other hand, we designed a new system. A single strand of DNA that is identical to the sequence of the signal chains mentioned in the above system but does not contain fluorescent groups is adsorbed to the microporous portal of mesoporous silicon by electrostatic attraction as a single strand of DNA, blocking the microporous portal and encapsulating the pre-equipped drug DOX inside the mesoporous silicon dioxide. Only when the linear long chain formed by the above-mentioned roll-ring amplification process occurs under the action of ATP, the carrier and controlled-release structure may hybridize with the single strand of gate DNA to break away from the surface of mesoporous silica and open the "door" on the micropore, thus releasing the drug. This design can achieve controlled release of the drug and effectively reduce the release of adriamycin. Similarly, in order to improve the selectivity of the system, the surface of mesoporous silica was modified with folic acid to realize the specific recognition of cancer cells overexpressing folic acid receptors, and then targeted drug delivery. The digestive effect and the side effects of anticancer drugs are reduced, which makes the detection method more sensitive, selective and feasible.
【学位授予单位】：山东师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R73-3;TQ460.1
本文编号：2203946
[Abstract]:Cancer diagnosis and treatment have attracted much attention in the field of biochemical analysis, and the methods involved are becoming more and more rich and innovative. Among them, functional nucleic acid nanosystems have shown good research and application prospects. Rolling Circle Amplification (RCA) and terminal metastasis reactions have led to the formation of a multifunctional DNA nanosphere system as a carrier of doxorubicin (DOX) for the specific recognition and targeted drug delivery of various cancer cells, and also for the determination of reducing sulfhydryl compounds in cells. Nanospheres have good biocompatibility and strong targeting selectivity, which can reduce the damage of normal cells and kill cancer cells at the same time. They have high sensitivity and good reproducibility for the detection of glutathione and other reductive sulfhydryl compounds. On the other hand, this paper is based on the roller amplification method. Furthermore, proximity ligation (PLA) was used to combine with graphene oxide and mesoporous silica to realize the detection of intracellular small molecules and the release of intracellular drugs. The main contents are as follows: 1. Detection of glutathione and targeted delivery of antitumor drug DOX by multifunctional magnetic DNA nanospheres based on DNA self-assembly technology and end-transfer reaction: Firstly, using the principle of DNA self-assembly technology, DNA nanoparticles were formed by rolling amplification method. Secondly, magnetic DNA nanospheres were introduced to form magnetic DNA nanospheres on the basis of synthesizing DNA nanospheres for easy isolation. DNA primer chains were modified to hybridize DNA single strands containing disulfide bonds, thus forming DNA nanospheres containing disulfide bonds. The nanospheres were able to achieve intracellular glutathione. Furthermore, the template chain involved in the roll-ring amplification process during the synthesis of DNA nanospheres was modified to contain the sgc8 aptamer sequence, which can specifically identify the over-expressed PTK7 protein on CEM cells and thus synthesize the aptamer containing sgc8 nucleic acid. Finally, on the basis of DNA nanospheres, we also transfer a number of adenine (A) bases to the linear long chain end formed by ring-rolling amplification, which can be hybridized with the polythymine (T) single strand modified with folic acid, by the action of terminal transferase. DNA nanospheres containing folic acid were formed by the reaction. These nanospheres can identify the cancer cells which overexpress folic acid receptors on the surface, such as HeLa cells, to achieve effective drug delivery. Drug release system: Proximity ligation (PLA) can bind two different single-stranded DNA molecules to the same protein recognition molecule, so that the tail of the two single strands of DNA can be closely spaced. Under the action of DNA ligase, the free 5'and 3'ends of the DNA chain react to form a ring-rolling amplification. Therefore, we have designed a DNA single strand containing ATP aptamer sequence as a link chain, a template chain which can complement the end of the link chain, and a signal chain which can partly resemble the sequence of the template chain. Once the system meets the ATP molecule, it can bind the aptamer chain of ATP to the ATP molecule, which results in the adjacent junction effect, and forms a circular DNA complex under the action of DNA ligase, thus completing the rolling amplification process. Long linear DNA single strands contain thousands of bases, which can bind to a large number of signal chains attached to graphene oxide and emit strong fluorescence. The ATP concentration can be measured by detecting the fluorescence. The detection signal is strong and the sensitivity is high. At the same time, in order to improve the selectivity of the system, we repair the surface of graphene oxide. Folic acid molecule can specifically bind to cancer cells with over-expression of folate receptor, such as HeLa cells. Compared with MCF-7 cells lacking folate receptor, it shows obvious high selectivity. The experimental results further illustrate the potential of this system in cancer diagnosis. On the other hand, we designed a new system. A single strand of DNA that is identical to the sequence of the signal chains mentioned in the above system but does not contain fluorescent groups is adsorbed to the microporous portal of mesoporous silicon by electrostatic attraction as a single strand of DNA, blocking the microporous portal and encapsulating the pre-equipped drug DOX inside the mesoporous silicon dioxide. Only when the linear long chain formed by the above-mentioned roll-ring amplification process occurs under the action of ATP, the carrier and controlled-release structure may hybridize with the single strand of gate DNA to break away from the surface of mesoporous silica and open the "door" on the micropore, thus releasing the drug. This design can achieve controlled release of the drug and effectively reduce the release of adriamycin. Similarly, in order to improve the selectivity of the system, the surface of mesoporous silica was modified with folic acid to realize the specific recognition of cancer cells overexpressing folic acid receptors, and then targeted drug delivery. The digestive effect and the side effects of anticancer drugs are reduced, which makes the detection method more sensitive, selective and feasible.
【学位授予单位】：山东师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R73-3;TQ460.1
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