【作者】 王佳；

【导师】 栗坤；

【作者基本信息】 燕山大学 ， 化学工程与技术， 2022， 硕士

【摘要】 结直肠癌(colorectal cancer,CRC)是一种常见的胃肠道恶性肿瘤,在癌症相关死亡的病因中排名第二,全球范围内每年有近两百万人成为确诊的结直肠癌患者。据2018年统计,我国结直肠癌的发病率居于第3位,且多数病人在确诊时已经接近晚期。由于结肠癌是早期不易检测,晚期手术也极易转移的癌症种类,开发合理的早期确诊手段成为治疗结肠癌的新思路。核酸适配体由人工体外合成的寡核苷酸片段,利用指数富集配体系统进化技术(即SELEX技术)从随机的寡核苷酸库池中筛选获得。核酸适配体可以通过其特殊的三维构象与目标分子特异性结合,作为具有高特异性和性质稳定的识别性小分子,在癌症早期检测中具有重要的发展前景。在本研究中,采用基于琼脂糖磁珠的筛选(Serum-SELEX)技术筛选了结肠癌血清的特异性适配体。为了获得高特异性的适配体,我们通过前期结肠癌血清正筛-正常人血清反筛步骤和后期结肠癌血清正筛-其他癌症血清反筛步骤循环的方式,经过16轮的筛选流程,完成后进行高通量测序。通过序列分析、碱基分布情况、二级结构和三级结构模拟,共选取4条候选适配体进行特性分析。通过对适配体序列的分析,胸腺嘧啶为适配体占比最高的主要碱基,其次是鸟嘌呤。这两者在大部分候选适配体中占据了随机序列域中约60%的比例。通过他们的二级结构预测可发现,候选适配体主要形成的结构是茎环和假结两种,这可能是适配体与血清中蛋白分子结合的优势结构域。通过进一步对候选适配体的三维结构进行模拟并用圆二色谱进行验证。候选适配体的亲和力使用q-PCR法验证,亲和力常数K_d均在10n M范围内,这表明了适配体具有优秀的亲和力。利用磁珠偶联法鉴定适配体与血清中蛋白分子的特异性情况,发现候选适配体APT-2具有良好的特异性,且此种方法的检出率为82.5%左右。进行ELASA试验对特异性结果进一步确认,验证了适配体APT-2的优异特异性。本研究最终筛选得到了结肠癌血清的特异性核酸适配体APT-2,并与结肠癌血清有适宜的亲和力(6.122nM)和优秀的特异性,表明该适配体在结肠癌的早期诊断手段上有着良好的前景。更多还原

【Abstract】 Colorectal cancer(CRC)is a common malignant tumor in the gastrointestinal tract,ranking third in cancer-related deaths.Every year,more than one million people in the world suffer from CRC.In 2018,the incidence of colorectal cancer in China ranked third,and the incidence is increasing year by year.As colonic cancer is difficult to detect in the early stage,and terminal operation is easy to transfer,the development of reasonable early diagnosis means becomes a new way to treat colonic cancer.Aptamers are synthetic oligonucleotide molecules screened from oligonucleotide libraries by exponential ligand enrichment system evolution(SELEX).Aptamers can bind specifically to target molecules through fixed spatial conformation.As small recognition molecules with high affinity,low cost and low immunogenicity,aptamers have important prospects in cancer detection.In this study,we use serum-selex(ser-selex)technique based on magnetic beads to screen serum specific aptamers for colon cancer.In order to obtain highly specific serum adaptors for colon cancer,normal serum,rectal cancer serum and gastric cancer serum were introduced as reverse screening targets.After 16 rounds of target reduction and replacement,screening was completed and high-throughput sequencing was performed.Through sequence analysis and comparison,secondary structure prediction and tertiary structure simulation,4 candidate aptamers were selected for characteristic analysis.The aptamer sequence analysis showed that thymine(T)and guanine(G)were the two bases with high proportion,which accounted for 60% of the 40 random sequence fields in the 4 aptamers.By predicting the secondary structure of the aptamer,we found that the aptamer mainly formed stem ring and pseudo junction structure,which may be the dominant domain of the aptamer.The aptamer APT-2 three-dimensional structure was simulated and verified by circular dichroism.The binding affinity between aptamer and target was measured by q-PCR,and the affinity constant Kd was all in the range of 10 n M,indicating that the aptamer had a high affinity with the target.The specificity of aptamer binding to target was identified by q-PCR method,and the results showed that aptamer APT-2 had high specificity and the positive detection was more than 80%.Furthermore,we also use ELASA to demonstrate the specificity of apt-2.In this study,serum specific aptamer of colon cancer was screened,especially apt-2,which showed good affinity(6.122±0.615 nM)and specificity(P<0.05)with a positive detection rate of more than 80%,indicating that this aptamer has great potential in the diagnosis of colon cancer.更多还原