早期胃腺癌原代细胞适配子的筛选与鉴定

胃腺癌是消化道最常见的恶性肿瘤之一，由于没有针对早期胃腺癌有效的诊断方法，目前胃腺癌手术治疗还主要针对中晚期患者，预后差. 本文应用cell-SELEX技术，筛选早期胃腺癌原代细胞的适配子，为早期胃腺癌的诊断提供新的思路. 从早期胃腺癌组织中分离得到早期胃腺癌原代细胞，应用体外合成全长88 bp中间含52 bp随机序列的单链DNA文库，通过对PCR扩增条件的优化，借助生物素-链霉亲和素磁珠系统，经cell-SELEX反复筛选，可获得针对早期胃腺癌原代细胞的特异性适配子.经12轮cell-SELEX筛选，ssDNA文库与早期胃腺癌原代细胞的亲和力由1 560上升到4 336，表明亲和力较高的适配子得到逐步富集. 经克隆和测序，应用软件分析可知，30个克隆子中编号为C17和C27的2个序列完全一致，具同源性，二级结构预测可知单链DNA形成不同的茎环结构可能是适配子与早期胃腺癌原代细胞作用的结构基础. 特异性分析显示，胃腺癌原代细胞组与正常胃粘膜上皮细胞、空白对照组之间荧光强度值差异非常显著（P＜001）；正常胃粘膜上皮细胞组与空白对照组之间差异不显著（P＞005）. 经亲和力测定，各适配子与早期胃腺癌原代细胞的解离系数达到nmol/L，具有很高的亲和力.利用cell-SELEX技术成功筛选到早期胃腺癌原代细胞的适配子，为胃腺癌的早期诊断与治疗药物靶点方面的研究奠定了实验基础.

Screening and Characterization of Aptamers in Early Gastric Adenocarcinoma Primary Cells

Gastric adenocarcinoma is one of the most common gastrointestinal malignant tumors. Due to the lack of efficient method for diagnosis in early gastric adenocarcinoma, doctors undergo operations on middle and terminal patients, which results in a poor prognosis. In this study, we employed cell-SELEX to screen aptamers in early gastric adenocarcinoma primary cells, which could provide new ideas for diagnosis of early gastric adenocacinoma. Early gastric adenocarcinoma primary cells were isolated from early gastric adenocarcinoma tissue, the ssDNA library with 88 bases in length containing a 52 base random sequence was synthesized in vitro, PCR conditions for ssDNA amplification were optimized, the products of PCR were dissociated by biotin-streptavidin magnetic beads system. After the repeative screenings, the aptamers specially bound to early gastric adenocarcinoma were obtained by cell-SELEX. After 12 cycles, the binding affinity between ssDNA library and early gastric adenocarcinoma has increased from 1560 to 4 336, indicating that aptamers with high affinity were enriched progressively. Then PCR products of the last round were cloned and sequenced. By analysis with DNAMAN70, we found that there were two sequences of C17 and C27 in the 30 clones utterly identified, which means a homology. Secondary structures prediction revealed that the stem-loop structures formed by ssDNA are possibly the action basis between aptamers and early gastric adenocarcinoma primary cells. In specificity, the fluorescence intensity of gastric adenocarcinoma primary cells were significantly different from that of normal gastric mucosal epithelium and the blank group (P<001). In affinity, the dissociation coefficient between aptamers and early gastric adenocaicinoma reached nmol level, indicating a high affinity. The aptamers specific to early gastric adenocarcinoma screened by cell-SELEX established the foundation for early diagnosis of gastric adenocarcinoma and the study of drug targets.