Selective Targeting to Glioma with Nucleic Acid Aptamers.
Selective Targeting to Glioma with Nucleic Acid Aptamers.
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DOI：
10.1371/journal.pone.0134957
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Shaw L
中科院分区：
文献类型：
--
作者：
Aptekar S;Arora M;Lawrence CL;Lea RW;Ashton K;Dawson T;Alder JE;Shaw L
恶性胶质瘤的特点是生长速度快，对周围脑组织侵袭性浸润能力强；因此，诊断和治疗困难，患者生存率差。适配体为活细胞和组织的体外成像提供了一种有前途和独特的技术，在恶性胶质瘤的临床诊断和治疗中具有潜在的光明前景。研究了两种DNA适体SA43和SA44在胶质瘤细胞和患者组织中的结合选择性、摄取能力和结合靶点。结果表明，SA43和SA44与U87MG靶细胞结合具有较强的亲和力（Kd分别为21.56±4.60 nM和21.11±3.30 nM）。流式细胞术定量分析显示，与非癌性和非胶质瘤细胞类型相比，适配体在U87MG和1321N1胶质瘤细胞中能够主动内化。共聚焦显微镜证实了细胞质中的染色，内质网、高尔基体和溶酶体标记物的共定位研究表明，内在化和区隔化在膜系统中。两种适体选择性结合ku70和ku80 DNA修复蛋白，通过细胞沉淀（AP）、质谱分析和Western blot确认。此外，石蜡包埋、福尔马林固定患者组织的细胞组织化学（AHC）染色显示，与非癌组织相比，SA43适体在胶质瘤组织（I级、II级、III级和IV级）中的结合选择性明显更高，而SA44对胶质瘤组织没有选择性。结果表明，SA43适体可以区分胶质瘤和非癌细胞和组织，因此在胶质瘤的组织学诊断中具有应用前景。
Malignant glioma is characterised by a rapid growth rate and high capacity for invasive infiltration to surrounding brain tissue; hence, diagnosis and treatment is difficult and patient survival is poor. Aptamers contribute a promising and unique technology for the in vitro imaging of live cells and tissues, with a potentially bright future in clinical diagnostics and therapeutics for malignant glioma. The binding selectivity, uptake capacity and binding target of two DNA aptamers, SA43 and SA44, were investigated in glioma cells and patient tissues. The binding assay showed that SA43 and SA44 bound with strong affinity (Kd, 21.56 ± 4.60 nM and Kd, 21.11 ± 3.30 nM respectively) to the target U87MG cells. Quantitative analysis by flow cytometry showed that the aptamers were able to actively internalise in U87MG and 1321N1 glioma cells compared to the non-cancerous and non-glioma cell types. Confocal microscopy confirmed staining in the cytoplasm, and co-localisation studies with endoplasmic reticulum, Golgi apparatus and lysosomal markers suggested internalisation and compartmentalisation within the endomembrane system. Both aptamers selectively bound to Ku 70 and Ku 80 DNA repair proteins as determined by aptoprecipitation (AP) followed by mass spectrometry analysis and confirmation by Western blot. In addition, aptohistochemical (AHC) staining on paraffin embedded, formalin fixed patient tissues revealed that the binding selectivity was significantly higher for SA43 aptamer in glioma tissues (grade I, II, III and IV) compared to the non-cancerous tissues, whereas SA44 did not show selectivity towards glioma tissues. The results indicate that SA43 aptamer can differentiate between glioma and non-cancerous cells and tissues and therefore, shows promise for histological diagnosis of glioma.