核酸适配体-银分子探针用于胶质瘤细胞膜及胞外基质中腱糖蛋白的原位高灵敏检测研究

Tenascin-C (TN-C) is synthesized primarily by glioma cells which is secreted to the extracellular matrix through the cell membrane. TN-C has been shown to play a significant role in the process of tumor initiation, growth, invasion and metastasis in gliomas. There is a close relationship between the TN-C expression and the tumor malignant grade. However, the conventional research methods are focused on the detection of the extracellular matrix accumulation, which could not realize detecting the localization and quantification of TN-C on the cell membrane in situ and therefore could not understand how the cell membrane regulates the protein accumulation in the matrix, which will prevent to accurately determine the relationship between the TN-C and the tumor progression..This study will design the aptamer-Ag molecular hybrid nanoprobes which have the specific targeting and stable fluorescence properties to express and detect TN-C. .With the help of a confocal microscope and the ion couple plasma mass spectra (ICP-MS),the TN-C locations and quantities on the cell membrane and the extracellular matrix can be calculated respectively. More importantly, this research can establish the corresponding relationship of the TN-C concentrations between the cell secretion and the accumulation in the extracellular matrix. The results will reveal how the glioma cells affect the protein content in the matrix by regulating the secretion of TN-C, which could provide a theoretical basis and technical support to reveal the mechanism that how the TN-C protein regulates the tumor deterioration.

腱糖蛋白主要由胶质细胞分泌，由细胞膜运输到胞外基质。在胶质细胞瘤发生、发展、侵袭及转移过程中起着十分重要的作用，与肿瘤的恶性度存在着密切关系。目前的研究方法只能检测胞外基质中腱糖蛋白的积累含量，无法原位高灵敏检测细胞膜上的分泌，无法了解细胞膜对于胞外基质中蛋白积累量的调控，因而无法准确判定腱糖蛋白与肿瘤恶化的关系。.本课题将构建基于核酸适配体的银分子复合纳米探针，利用探针来表达检测腱糖蛋白。借助核酸适配体的靶向定位及银分子探针的稳定荧光特性，选用三维立体活细胞培养体系模拟肿瘤组织微环境，利用激光共聚焦显微成像技术结合电感耦合等离子体质谱分析方法，高灵敏度原位检测胶质瘤细胞的腱糖蛋白:分别研究膜表面和胞外基质中蛋白的空间分布和表达浓度，建立细胞膜分泌蛋白与基质积累蛋白的相应关系，以此来研究胶质瘤细胞如何通过调控腱糖蛋白的分泌进而影响基质中蛋白含量，为揭示腱糖蛋白调控肿瘤的恶化提供技术支持。

在生命体系中蛋白发挥着重要的作用，进一步的研究表明，细胞膜表面蛋白的分布和含量的改变与肿瘤的发生发展、侵袭及恶性程度密切相关。然而传统分析方法对于膜蛋白的分布研究多利用荧光抗体共定位方法，无法实现对于活细胞内蛋白的实时成像观察；同时， ELISA、双向凝胶电泳以及同位素标记的质谱技术等定量蛋白的技术需要建立标准曲线，并且需要提取细胞裂解产物从而达到间接定量的目的，无法实现活细胞原位检测。本项目借助核酸适配体的靶向定位及银团簇的稳定荧光特性，构建了能够特异识别细胞膜表面蛋白的核酸适配体-银分子团簇探针，利用激光共聚焦成像技术结合质谱分析技术实现了对于蛋白的分布和含量进行实时原位高灵敏的检测。借助核酸适配体的不同靶向识别性，该项目构建了系列蛋白分析模型，这一研究有助于发展细胞诊疗技术。