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目的采用指数级富集配体的系统进化(SELEX)技术筛选高转移肝癌细胞HCCLM3的ssDNA适配子。方法构建长度为71 nt的随机文库,中间为25 bp的随机序列,通过12轮反复的SELEX筛选获得特异性结合HCCLM3细胞的ssDNA适配子,并初步测定筛选的适配子与细胞的亲和力,对结合力高的适配子进行克隆测序,用RNA structure 5.3分析适配子序列的二级结构。结果经过12轮正向筛选及6轮低转移肝癌细胞MHCC97-L的反筛后,获得了与HCCLM3细胞具有高亲和力的ssDNA适配子;将第12轮ssDNA经克隆测序获得10条测序结果,其中有4条序列一致,另外有2条序列也一致;二级结构预测可知ssDNA适配子二级结构主要以“发夹型”构型为主。结论成功筛选获得了高亲和力的转移肝癌细胞HCCLM3 ssDNA适配子,为实现适配子靶向性治疗奠定了一定的理论基础。
OBJECTIVE: To screen ssDNA aptamers of HCCLM3 cells with high metastatic potential using SELEX (exponential enrichment ligand) method. Methods A random sequence of 71 nt was constructed with a 25 bp random sequence in the middle. The ssDNA aptamers specifically binding to HCCLM3 cells were obtained by 12 rounds of repeated SELEX screening. The affinity of the selected aptamers to cells was determined, Aptamers with high binding capacity were cloned and sequenced, and the secondary structure of the aptamer sequence was analyzed by RNA structure 5.3. Results After 12 rounds of positive screening and 6 rounds of reverse transfection of MHCC97-L cells with low metastatic potential, ssDNA aptamers with high affinity to HCCLM3 cells were obtained. Ten rounds of sequencing results were obtained by cloning and sequencing of round 12 ssDNA. Among them, four sequences were identical and the other two sequences were also consistent. The secondary structure prediction indicated that the secondary structure of ssDNA aptamers mainly consisted of “hairpin ” configuration. Conclusion Successful screening of HCCLM3 ssDNA aptamers with high affinity for hepatocellular carcinoma cells has laid a theoretical foundation for the targeted therapy of aptamers.