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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.
Gastric adenocarcinoma is one of the most common malignant tumors of the digestive tract, and because there is no effective diagnostic method for early gastric adenocarcinoma, the current surgical treatment of gastric adenocarcinoma is also mainly aimed at patients with advanced stage and poor prognosis.This article uses cell-SELEX technique to screen early gastric adenocarcinoma The aptamers of primary cells provide a new idea for the diagnosis of early gastric adenocarcinoma.Preliminary primary gastric adenocarcinoma cells were isolated from early gastric adenocarcinoma tissues and were synthesized by in vitro synthesis of a single chain DNA library, through the optimization of PCR amplification conditions, with biotin-streptavidin magnetic beads system, repeated screening by cell-SELEX can be obtained for early gastric adenocarcinoma of the primary cell specific aptamers .12 Round cell-SELEX screening, the affinity of ssDNA library with primary gastric adenocarcinoma cells increased from 1 560 to 4 336, indicating that the higher affinity aptamers were gradually enriched.Through cloning and sequencing, software analysis showed that 30 The two sequences of clone C17 and C27 were identical and homologous. The secondary structure predicts that the formation of different stem-loop structures of single-stranded DNA may be the structure of the aptamer acting on the primary cells of early gastric adenocarcinoma Specificity analysis showed that there was significant difference in fluorescence intensity between primary gastric adenocarcinoma cells and normal gastric mucosal epithelial cells and blank control group (P <0.01), but there was no difference between normal gastric mucosal epithelial cells and blank control group (P> 0.05) .According to the affinity assay, the dissociation coefficient of each aptamer and the primary gastric adenocarcinoma cells reached nmol / L with high affinity.The cell-SELEX technique was used to screen the early gastric adenocarcinoma Generation of cell aptamers, for the early diagnosis of gastric adenocarcinoma and treatment of drug targets in the study laid the foundation for the experiment.