【作者】 王成龙；

【导师】 王会信； 邵宁生；

【作者基本信息】 中国人民解放军军事医学科学院 ， 生物化学与分子生物学， 2002， 博士

【摘要】 恶性肿瘤是目前人类死亡的主要原因之一。人类对肿瘤的早期诊断和有效治疗缺乏特异性是恶性肿瘤对人类生存产生巨大威胁的主要原因。目前所发现并用于早期临床诊断的肿瘤标志物与正常细胞相比还没有质的区别,只是量的不同,缺乏真正的特异性;手术切除、放疗和化疗的联合应用是目前临床上治疗肿瘤的主要手段,虽然手术切除是去除实体瘤组织的有效方法,但在切除肿瘤组织的同时把一部分正常组织也扩大切除了,对人体创伤大,并且还有复发和转移的危险,术后仍需放疗和化疗,但是,不论是放射性同位素还是化疗药物对于肿瘤细胞和正常细胞都不加区分的一起杀死,因此在治疗肿瘤的同时也对人体造成了极大的损害,并且由于不能使用较大剂量而严重影响放疗和化疗的效果。当前恶性肿瘤早期诊断和有效治疗方面存在的诸多不利因素的一个根本原因是人们对正常细胞和恶性肿瘤细胞之间差异的理解缺乏足够的知识。 SELEX(Systematic evolution of ligands by exponential enrichment)技术是20世纪90年代发展起来的一种生物文库技术,该技术的目的就是利用人工合成的随机寡核苷酸文库筛选某一靶分子的特异寡核苷酸配基。由于SELEX技术具有可筛选的靶分子范围非常广,理论上能筛选已知的所有分子;筛选得到的配基亲合力和特异性极强,远高于其他任何类型的配基;操作简便,筛选周期短等优点,SELEX技术在临床诊断和疾病治疗方面具有广阔的应用前景。 本研究将消减杂交的原理引入SELEX筛选,分别以分化前后PC12细胞为消减靶子和筛选靶子,建立消减SELEX技术,并筛选获得特异识别已分化PC12细胞而不识别未分化PC12细胞的单链DNA(single stranded DNA,ssDNA)配基。期望能够为临床肿瘤诊断和治疗提供新的、实用的技术方法。 为了建立以完整细胞为靶子的消减SELEX技术,我们首先建立以单一蛋白为靶子,用随机ssDNA文库进行SELEX筛选的方法,为建立以完整细胞为靶子的消减SELEX技术提供技术准备。研究结果表明:同样大小的ssDNA和双链DNA(double-stranded DNA,dsDNA)在7M尿素12%变性聚丙烯酰胺凝胶电泳更多还原

【Abstract】 Malignancy is one of the main reasons of people death at present, the main reasons of which are the lack of specificity in inchoate diagnose and treatment availably. Compared with the normal cells, there is no qualitative but only quantitative difference for tumor marker found and used in inchoate diagnose. Until now, the combination of operation resection radiative treatment and chemical drug treatment is still the first consideration in tumor treatment. There are lots of side effects for these treatments. Operation resection will cause great wound. Radiation and chemical drugs will kill tumor cells and normal cells without distinguishing. So it is important to find a way to search new tumor markers.Systematic evolution of ligands by exponential enrichment (SELEX) is a combinatorial chemistry methodology for in vitro selection of specific aptamers — oligonucleotide ligands. Considerable progress has been achieved in this field since the establishment of SELEX in 1990. Aptamers specific for a wide variety of targets have been selected, demonstrating that aptamers for almost every target can now be obtained. Nucleic acid aptamers selected by SELEX show high specificity for their targets and could distinguish between homologous proteins or nearly identical low molecular weight compounds. SELEX has been widely used in searching for new oligonucleotides and has many potential applications in basic research, diagnostic and therapeutic purposes.In this work, subtractive SELEX, a new SELEX procedure incorporating the principle of subtraction, was established and successfully used in selecting of ssDNA aptamers, which could recognize differentiated PC 12 cells from undifferentiated PC 12 cells specifically.For establishing of subtractive SELEX method targeted the whole cell, it’s necessary to investigate firstly the feasibility that random ssDNA pool was used to perform SELEX selection targeted pure protein. We found that the eletrophoresis action was different in the 10% of denatured polyacrylamide (0.5% of bisacrylamide) gel containing 7M of urea between the same random ssDNA pool and random dsDNApool, the eletrophoresis rate of ssDNA pool was slower than that of dsDNA pool because ssDNA could form complex steric structure. Based on this, firstly we amplified ssDNA and dsDNA using asymmetric PCR method, then ssDNA product was purified with 10% of denatured polyacrylamide (0.5% of bisacrylamide) gel containing 7M of urea for the next round of SELEX selection. The pool capability of the random ssDNA pool used in this work was determined by cloning and the nucleotide sequering. The four bases in the central random region had about equal distribution, and A+T and C+G were about equal in each of the central random position except exceptional position. Then we carried out SELEX process to select streptavidin ssDNA aptamers with this random ssDNA pool. After 12 round selections, we found that the aptamers of final round pool had been enriched by Flow cytometry. The structure analyzed by software RNAstructure 3.5 showed a single stem-loop secondary structure. We approve streptavidin — specific ssDNA aptamers using biotin. It is feasible to determine the enrichment and specificity of the beads or cells using FITC (fluorescein isothiocyanat, FITC) -labeled aptamers by Flow cytometry.Incorporating the principle of subtraction, we established subtractive SELEX method targeted the whole cell. Subtractive selection was performed by firstly incubating ssDNA pools with undifferentiated PC 12 cells in selection buffer prior to each round of selection. Partitioning of bound and unbound ssDNA was done by centrifugation. The subtracted SELEX pools were incubated with differentiated PC 12 cells in selection buffer, cell-bound aptamers were eluted by elution, and were amplified by PCR. PCR products were used as templates to prepare ssDNA by asymmetric PCR for the next round of SELEX. After 6 round of subtractive SELEX selection, Radiolabeled aptamer binding assays results showed that the amount of radioactivity bound to differentiated PC 12 cells was higher than that to undifferentiated PC 12 cells in the 3rd and the 6th round. Flow cytometry with FITC-labeled aptamers showed a slow but steady increase of binding to differentiated PC 12 cells from round 2 to round 6. There was no significant change for the fluorescence intensity bound to undifferentiated PC 12 cells. Compared to the starting pools, G-base content in round 6 became higher, which had about equal distribution of all the four bases. Individual cloned aptamers were evaluated by flow cytometry. All of the 23 sequences showed specific binding to differentiated PC12 cells. No binding to undifferentiated PC 12 cells was observed. Aptamers from the starting pool did not bind to either cell. Among 23 ligands tested, aptamer 17 displayed the most intensive fluorescence binding to differentiated PC 12 cells. Two separate peaks of nearly equal counts resulted from aptamer17 binding, presumably one of the differentiated and theother parental PC 12 cells. These results indicate that aptamer 17 specifically recognizes the differentiated PC 12 cells from a mixture of various cellsThe completion of the aptamer selection process typically yields a high affinity and specific antagonist of the targeted protein. Several postselection optimization steps general must be performed to translate a molecule from an in vitro antagonist to a molecule that can be tested for pharmacologic effect in animals or used in vivo in target-validation studies. Chemical synthesis is required to produce the quantities of aptamer needed for most if not all in vivo experiments. Currently, for efficient and cost-effective chemical synthesis, the size of an aptamer must be reduced to fewer than 45 nucleotides, from a starting molecule of approximately 80 to 100 nucleotides. We truncate aptamer 17 from subtractive SELEX to 25 nucleotides (AP17-25) 38 nucleotides (AP17-38)and 48 nucleotides (API7-48) respectively by cutting defined primer-binding sequences partially or overall. AP17-25 AP17-38 and AP17-48 showed specific binding to differentiated PC 12 cells. No binding to undifferentiated PC 12 cells was observed using both Flow cytometry and fluorescent microscope. Two separate peaks of nearly equal counts resulted from API7-38 binding using Flow cytometry. AP17-38 showed specific binding to cytoplasm of differentiated PC12 cells by laser cofocalize microscope.Above all, we have established subtractive SELEX, and selected successfully ssDNA aptamers with the specificity to distinguish closely related cells. If the specific oligonucleotide ligands could be found to distinguish a certain type of cell from others, e.g. tumor cells from normal cells or cells of different developmental stages, these aptamers might be of important usefulness in basic research and clinical tumor diagnosis. This method may be useful in tumor diagnosis and therapy, and studies of cell differentiation. It also can be applied in finding novel proteins if combined with functional cloning.更多还原