单点突变蛋白特异的RNA适配体筛选及功能研究
发布时间：2019-06-11 13:19
【摘要】：p53蛋白,也被认为是肿瘤的抑制蛋白。自从发现以来,被证实参与细胞周期调控,激活DNA修复、维持基因组稳定及促进细胞凋亡。在超过50%的人类的癌症中都出现p53功能缺失,其中就包括单位点和多位点的突变及结构的扭曲。同样也有越来越多的研究证实,p53蛋白的突变,使得其空间构象发生变化,从而失去对细胞周期调控与促进细胞凋亡的重要生物学功能,与此同时,还会得到新的功能(Gain-of-Function, GOF),如促进细胞转移、侵染等,由抑癌基因变为癌基因。目前越来越多的研究组开始关注p53蛋白突变体的研究,其中就包括促进突变体的回复突变及抑制其生物学功能。此项研究对于癌症的靶点治疗具有重要的生物学研究与医学价值。 核酸适配体(aptamer)是一段短的单链核酸分子,其特点是能够根据结构特异性针对性地地结合多种靶标分子,如蛋白质、小分子、糖、脂肪等。它是由一个叫SELEX (Systematic Evolution of Ligand Exponential enrichment)的体外筛选进程得到的。SELEX技术自从1990年由三个研究组同时发明并已广泛地运用到诸如核糖体开关、靶向治疗等生物及医学应用。于是我们有理由相信,通过此筛选方法,我们也可以筛选到对诸如p53突变体有高亲和力和特异性的核酸适配体。 目前,还未见研究组筛选到p53突变体适配体的报导。在本论文中,我们的首要研究目的就是能够筛选到p53R175H的适配体。由于此突变型与野生型的唯一区别只在于第175位的氨基酸突变,所以我们对传统的SELEX筛选过程进行了改进,名为差别竞争SELEX筛选,将p53野生型与突变型蛋白质分别经过固相偶联,同时投入体系中,竞争性地结合库分子,最大限度地增加筛选特异性与差异性。经过5轮的差别竞争SELEX筛选,我们得到了21个p53R175H蛋白的候选适配体。通过亲和力检测,适配体p53R175H-APT与p53R175H蛋白的亲和力大大超过对于野生型p53的亲和力,并且在凝胶迁移实验中我们最终确定p53R175H-APT能够在体外和p53R175结合。所以我们最终确定的p53R175H-APT作为我们后续研究的对象。 尽管在亲和力检测中,p53R175H-APT对于p53突变体有着相对于野生型p53较强的亲和力,但其细胞学效应还有待研究。在本论文中,我们试图阐述筛选到的适配体P53R175H-APT在细胞中的功能。首先,我们必须确定P53R175H-APT在其靶标的选择上具有严格的特异性。在含有p53野生型的HEK293T和HeLa细胞中,通过将p53R175H-APT转染入上述两种细胞中,无论从表型还是从细胞生长于凋亡的情况看,均没有发现与对照组有显著性差异。同时,在只含有p53的另一个突变体p53R273H的H1299细胞中,经过转染p53R175H-APT,相对于对照组我们依然没有观察到明显的差别。在完成了以上实验证实p53R175H-APT对于含有p53野生型与p53其他突变体的细胞没有显著性作用之后,我们将此适配体构建到质粒并转染入H1299-p53R175H稳转细胞系中,我们发现,经过p53R175H-APT处理的细胞,细胞增殖得到明显的抑制,细胞的死亡明显增多。而对于含有p53野生型的细胞,则没有此现象。侧面说明此RNA适配体可以特异性地识别p53R175H并显著影响细胞增殖与细胞凋亡。为了更加确定产生如此明显的生物学差别与p53突变体相关,我们通过p53的免疫沉淀证实了在细胞中,p53R175H-APT与p53突变体结合的确大于IgG和scramble序列对p53突变体的结合能力。与此同时,通过克隆形成实验与soft agar实验我们证明了通过此适配体处理的细胞,其恶性程度与生长能力也有明显降低。由于很多p53的突变属于功能获得型突变,肿瘤细胞的迁移能力会不同程度地提高。为此我们设计了Transwell迁移实验和wound healing实验,证实经过此适配体处理过的细胞其迁移能力得到明显降低。进一步,在分子水平上,p53R175H-APT处理的细胞表现出更像p53野生型的调控方式,多个p53相互作用基因均有激活,一定程度上说明此适配体可使p53突变体的功能回复。同时,我们通过免疫荧光更加直观地证明了,经过p53R175H-APT处理过的p53突变体,能够从构象上使其回复成p53野生型。 为了从更高水平验证此效应可以运用到实体瘤的治疗中,我们建立了裸鼠肿瘤模型,并将适配体通过实体瘤注射与静脉注射两种方法进行治疗。相对于对照组,适配体组的裸鼠肿瘤明显减小并维持较低水平,甚至消失。通过对实体瘤切片的TUNEL凋亡检测染色,我们发现在适配体治疗组中的凋亡细胞明显多于对照组。更加证实了p53R175H-APT可以通过p53R175H作用于肿瘤细胞,促进肿瘤细胞的凋亡、降低细胞生长能力、限制肿瘤细胞的迁移及具有一定程度的临床潜力。 综上所述,本论文的研究成果阐述了一个新的SELEX筛选方法,并通过此方法成功筛选出与野生型p53只有一个碱基差异的p53R175H的适配体p53R175H-APT。体外实验证实其可以抑制肿瘤细胞生长并促进其凋亡,体内实验进一步证实了其潜在的临床价值,对于肿瘤靶向治疗提供新的思路。
[Abstract]:The p53 protein is also thought to be a tumor suppressor protein. Since the discovery, it has been demonstrated to be involved in cell cycle regulation, to activate DNA repair, to maintain genomic stability, and to promote cell apoptosis. P53 function deletions occur in more than 50% of human cancers, including mutation and structural distortion of the unit and multi-site points. In the same way, more and more studies have confirmed that the mutation of p53 protein causes the spatial conformation of the p53 protein to change, thus losing the important biological function of regulating and regulating the cell cycle and promoting the cell apoptosis, and at the same time, a new function (Gain-of-Function, GOF) can be obtained, such as promoting cell transfer, Infeiting and the like, the tumor suppressor gene becomes an oncogene. At present, more and more research groups have started to focus on the study of p53 protein mutants, including the promotion of the reverse mutation of the mutant and the inhibition of its biological function. The study has important biological and medical value for the treatment of cancer. The nucleic acid aptamer is a short-stranded nucleic acid molecule, which is characterized in that a plurality of target molecules, such as proteins, small molecules, sugar, fat, and so on. It is obtained by an in vitro screening process called SELEX The. SELEX technology has been invented by the three research groups in 1990 and has been widely used in the fields of biology and medicine, such as the ribosome switch, targeted therapy, etc. With this, we have reason to believe that by this screening method, we can also screen for nucleic acid adaptation, such as the high affinity and specificity of the p53 mutant in addition, it has not been found in that study group to screen the variant of the p53 mutant In this paper, our primary purpose is to be able to filter to p53R175H As the only difference between this mutant and the wild type is the amino acid mutation at position 175, we have improved the traditional SELEX screening process, called the differential competitive SELEX screening, and the p53 wild-type and the mutant protein are coupled through a solid phase, respectively, at the same time In the system, the library molecules are bound in a competitive way, and the screening specificity is increased to the maximum extent. The difference. After a 5-wheel differential competitive SELEX screening, we got a candidate for 21 p53R175H proteins The affinity of the aptamer p53R175H-APT with the p53R175H protein greatly exceeded the affinity for wild-type p53 by affinity detection, and in the gel migration experiment we finally determined that the p53R175H-APT was capable of in vitro and p53R17 5. So our final p53R175H-APT is our follow-up study The p53R175H-APT has a strong affinity for p53 mutants with respect to wild-type p53, but its cytologic effect Also to study. In this paper, we try to set out the filter to which the aptamer P53R175H-APT is fine The function in the cell. First, we must determine that the P53R175H-APT has a strict selection of its target The specificity of the cells. In HEK293T and HeLa cells containing the p53 wild type, none of the two cells was found by transfecting p53R175H-APT into the two cells, either from the phenotype or from the cell growth to the apoptosis. At the same time, in the H1299 cells of another mutant p53R273H containing only p53, the p53R175H-APT was transfected and we were still not observed with respect to the control group There is a clear difference. After the above experiments have been completed to confirm that p53R175H-APT has no significant effect on the cells containing the p53 wild-type and p53 other mutants, we construct the aptamer into the plasmid and transfect into the H1299-p53R175H stable-transfer cell line, and we have found that the cell proliferation obtained through the p53R175H-APT treatment Obvious inhibition, cell death There is a significant increase in death, whereas for cells containing wild-type p53, There is no such phenomenon. The side shows that this RNA aptamer can specifically recognize p53R175H and significantly affect cell proliferation In order to make it more determined that such a significant biological difference is associated with a p53 mutant, the immunoprecipitation of p53 confirmed that the combination of p53R175H-APT with the p53 mutant is indeed greater than that of the IgG and scabble sequence to the p53 mutant At the same time, by cloning and forming an experiment with soft agar, we demonstrated that the cells treated with this aptamer, the degree of malignancy and the ability to grow, There is a significant reduction in the ability of the tumor cells to migrate due to a variety of p53 mutations that are functional to type mutations. To this end, we designed the Transwell Migration Experiment and the Sound Rheing experiment to confirm that the cells treated by this aptamer have the ability to migrate. Further, at the molecular level, the cells treated with p53R175H-APT exhibited more p53 wild-type control, and many of the p53-interacting genes were activated to a certain extent that the aptamer could be used to make the p53 mutant The functional response of the p53R175H-APT, which was treated by p53R175H-APT, was more intuitively demonstrated by immunofluorescence. 53 Wild-type. In order to verify this effect from a higher level that can be applied to the treatment of solid tumors, we established a nude mouse tumor model and injected the adapter through solid tumor injection and intravenous injection. In contrast to the control group, the tumor of the nude mice of the aptamer group was significantly reduced and kept low The level, or even the disappearance, was detected by the TUNEL apoptosis test of the solid tumor section, and we found that the apoptotic cells in the aptamer treatment group It is more evident that p53R175H-APT can act on the tumor cells through p53R175H, promote the apoptosis of the tumor cells, reduce the cell growth ability, limit the migration of the tumor cells, In conclusion, the results of this paper describe a new SELEX screening method and successfully screened the adaptor p53 of p53R175H with only one base difference with the wild-type p53 by this method. R175H-APT. In vitro experiments confirm that it can inhibit the growth of tumor cells and promote its apoptosis. In vivo, the potential clinical value is further confirmed, and for tumor target
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R73-36
[Abstract]:The p53 protein is also thought to be a tumor suppressor protein. Since the discovery, it has been demonstrated to be involved in cell cycle regulation, to activate DNA repair, to maintain genomic stability, and to promote cell apoptosis. P53 function deletions occur in more than 50% of human cancers, including mutation and structural distortion of the unit and multi-site points. In the same way, more and more studies have confirmed that the mutation of p53 protein causes the spatial conformation of the p53 protein to change, thus losing the important biological function of regulating and regulating the cell cycle and promoting the cell apoptosis, and at the same time, a new function (Gain-of-Function, GOF) can be obtained, such as promoting cell transfer, Infeiting and the like, the tumor suppressor gene becomes an oncogene. At present, more and more research groups have started to focus on the study of p53 protein mutants, including the promotion of the reverse mutation of the mutant and the inhibition of its biological function. The study has important biological and medical value for the treatment of cancer. The nucleic acid aptamer is a short-stranded nucleic acid molecule, which is characterized in that a plurality of target molecules, such as proteins, small molecules, sugar, fat, and so on. It is obtained by an in vitro screening process called SELEX The. SELEX technology has been invented by the three research groups in 1990 and has been widely used in the fields of biology and medicine, such as the ribosome switch, targeted therapy, etc. With this, we have reason to believe that by this screening method, we can also screen for nucleic acid adaptation, such as the high affinity and specificity of the p53 mutant in addition, it has not been found in that study group to screen the variant of the p53 mutant In this paper, our primary purpose is to be able to filter to p53R175H As the only difference between this mutant and the wild type is the amino acid mutation at position 175, we have improved the traditional SELEX screening process, called the differential competitive SELEX screening, and the p53 wild-type and the mutant protein are coupled through a solid phase, respectively, at the same time In the system, the library molecules are bound in a competitive way, and the screening specificity is increased to the maximum extent. The difference. After a 5-wheel differential competitive SELEX screening, we got a candidate for 21 p53R175H proteins The affinity of the aptamer p53R175H-APT with the p53R175H protein greatly exceeded the affinity for wild-type p53 by affinity detection, and in the gel migration experiment we finally determined that the p53R175H-APT was capable of in vitro and p53R17 5. So our final p53R175H-APT is our follow-up study The p53R175H-APT has a strong affinity for p53 mutants with respect to wild-type p53, but its cytologic effect Also to study. In this paper, we try to set out the filter to which the aptamer P53R175H-APT is fine The function in the cell. First, we must determine that the P53R175H-APT has a strict selection of its target The specificity of the cells. In HEK293T and HeLa cells containing the p53 wild type, none of the two cells was found by transfecting p53R175H-APT into the two cells, either from the phenotype or from the cell growth to the apoptosis. At the same time, in the H1299 cells of another mutant p53R273H containing only p53, the p53R175H-APT was transfected and we were still not observed with respect to the control group There is a clear difference. After the above experiments have been completed to confirm that p53R175H-APT has no significant effect on the cells containing the p53 wild-type and p53 other mutants, we construct the aptamer into the plasmid and transfect into the H1299-p53R175H stable-transfer cell line, and we have found that the cell proliferation obtained through the p53R175H-APT treatment Obvious inhibition, cell death There is a significant increase in death, whereas for cells containing wild-type p53, There is no such phenomenon. The side shows that this RNA aptamer can specifically recognize p53R175H and significantly affect cell proliferation In order to make it more determined that such a significant biological difference is associated with a p53 mutant, the immunoprecipitation of p53 confirmed that the combination of p53R175H-APT with the p53 mutant is indeed greater than that of the IgG and scabble sequence to the p53 mutant At the same time, by cloning and forming an experiment with soft agar, we demonstrated that the cells treated with this aptamer, the degree of malignancy and the ability to grow, There is a significant reduction in the ability of the tumor cells to migrate due to a variety of p53 mutations that are functional to type mutations. To this end, we designed the Transwell Migration Experiment and the Sound Rheing experiment to confirm that the cells treated by this aptamer have the ability to migrate. Further, at the molecular level, the cells treated with p53R175H-APT exhibited more p53 wild-type control, and many of the p53-interacting genes were activated to a certain extent that the aptamer could be used to make the p53 mutant The functional response of the p53R175H-APT, which was treated by p53R175H-APT, was more intuitively demonstrated by immunofluorescence. 53 Wild-type. In order to verify this effect from a higher level that can be applied to the treatment of solid tumors, we established a nude mouse tumor model and injected the adapter through solid tumor injection and intravenous injection. In contrast to the control group, the tumor of the nude mice of the aptamer group was significantly reduced and kept low The level, or even the disappearance, was detected by the TUNEL apoptosis test of the solid tumor section, and we found that the apoptotic cells in the aptamer treatment group It is more evident that p53R175H-APT can act on the tumor cells through p53R175H, promote the apoptosis of the tumor cells, reduce the cell growth ability, limit the migration of the tumor cells, In conclusion, the results of this paper describe a new SELEX screening method and successfully screened the adaptor p53 of p53R175H with only one base difference with the wild-type p53 by this method. R175H-APT. In vitro experiments confirm that it can inhibit the growth of tumor cells and promote its apoptosis. In vivo, the potential clinical value is further confirmed, and for tumor target
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R73-36
【共引文献】
相关期刊论文 前10条
1 曾冉;况建国;;胶质瘤发病机制的研究进展[J];广东医学;2013年06期
2 王国玉;倪晶;;纳米磁球偶联重组p53蛋白检测p53自身抗体的新颖方法[J];放射免疫学杂志;2013年05期
3 刘燕;于晓峰;邹健;;14-3-3σ蛋白对肿瘤细胞的调控机制[J];国际消化病杂志;2013年06期
4 吴鹏飞;;抑癌基因p53与原发性肝癌的研究进展[J];河南医学研究;2010年03期
5 陈青;邬黎青;;p53基因和乳腺癌的相关性研究进展[J];实验与检验医学;2011年01期
6 MENG LingFeng;CHEN Liang;LI ZhaoYong;WU ZhengXing;SHAN Ge;;Environmental RNA interference in animals[J];Chinese Science Bulletin;2013年35期
7 林艳端;申锷;白文坤;南淑良;胡兵;;低频低功率超声联合微泡对DU145细胞及PC3细胞早期凋亡的影响[J];临床超声医学杂志;2014年01期
8 陈宏健;谢松;李理想;柳峰松;;一种基于干扰肌动蛋白基因导致方形网纹n，
本文编号：2497214
本文链接：https://www.wllwen.com/yixuelunwen/zlx/2497214.html
