文献：

Study on apoptosis of tumor cells induced bynucleolin cross-linking on cell surface

文献链接：

https://xueshu.baidu.com/usercenter/paper/show?paperid=105j0ag0em2p0gx0d1480pn03v328176&site=xueshu_se&hitarticle=1

作者：程凤

原文摘要：

Traditional tumor clearance methods suffer from poor targeting and high recurrence rates. Using external stimuli to induce the aggregation of cell surface receptors, activate intracellular apoptotic signaling pathways, reconstruct the apoptotic signaling system of tumor cells, and trigger tumor cell apoptosis has become a means. At present, the cell membrane receptors studied do not have universality, and the materials used for receptor aggregation regulation have problems such as short action time, poor stability, and easy endocytosis. Therefore, a strategy based on overexpression of nucleolin on the surface of tumor cells was developed to induce apoptosis and achieve in vitro and in vivo anti tumor effects. By constructing a multifunctional probe GC chol act CDNA and anchoring it to the cell membrane for a long time, the relative movement of membrane receptors is restricted, and the formation of cross-linked receptors is induced, achieving real-time imaging of membrane proteins and other functions. The preparation method is as follows:

Using ethylene glycol chitosan (GC) as the polymer skeleton, an intermediate product GC col pt was formed by amide reaction between Cy3 aptamer labeled with side chain fluorescent dye Cy3 and cholesterol. In order to reduce false positive signals caused by non-specific adsorption of probes on cell membranes, a short complementary sequence cDNA labeled with quencher BHQ2 was introduced. By hybridizing with the aptamer to form a double stranded structure, a flexible "multi gripper" complex GC chol act cDNA was constructed. The results of Cy3 fluorescence quenching, gel electrophoresis, hydrated particle size and atomic force microscopy showed that GC chol apt cDNA was successfully synthesized, and its structure was spherical with a particle size of about 500 nm. The fluorescence co localization imaging experiment of cell membrane dyes and nucleolin antibodies showed that the probe can specifically target the nucleolin on the surface of tumor cells, causing cDNA to compete and Cy3 fluorescence to recover, achieving accurate and specific targeted imaging of tumor cell surface nucleolin. More importantly, the structure is anchored to the cell membrane through the affinity of aptamers and the hydrophobicity of cholesterol, effectively inhibiting cell endocytosis and remaining stable on the cell surface for a long time. The results of flow cytometry and fluorescence confocal imaging showed that the affinity of GC chol pt to cells increased by nearly 100 times, and it could stably anchor on the cell membrane for up to 6 hours, providing a powerful tool for long-term cell membrane imaging.

传统的tumour清除手段存在靶向性差、复发率高等问题。利用外部刺激诱导细胞表面受体相互聚集,激活细胞内Apoptosis 信号通路，重建tumour细胞的Apoptosis 信号传递系统，触发tumour细胞Apoptosis 成为一种手段。目前所研究的细胞膜受体不具备通用性，且用于受体聚集调控的材料作用时间短、稳定性差、易内吞等问题。因此，开发了一种基于tumour细胞表面过表达的核仁素交联诱导细胞Apoptosis 的策略实现体内外抗tumour。通过构建探针乙二醇壳聚糖-胆固醇-Cy3-核酸适配体（GC-chol-apt-CDNA），长时间锚定在细胞膜上，限制膜受体相对运动，并诱导受体形成交联状态，实现集膜蛋白实时成像等作用。制备方法如下：

图：(a)GC-chol-apt-cDNA 的合成示意图

以乙二醇壳聚糖(glycolchitosan,GC)为高分子骨架，通过酰胺反应在侧链接枝荧光染料Cy3标记的核酸适配体(Cy3-aptamer)和胆固醇(cholesterol)，形成了中间产物 GC-chol-apt。为了降低因探针在细胞膜上的非特异性吸附作用带来的假阳性信号,引入了猝灭剂 BHQ2 标记的短链互补序列cDNA，通过与 aptamer 部分杂交形成双链结构,构建了一种柔性“多抓手”复合物 GC-chol-apt-cDNA。通过 Cy3 荧光猝灭、凝胶电泳、水合粒径及原子力显微镜等手段表明实验成功合成了GC-chol-apt-cDNA,其结构呈球形,粒径约为 500nm。

细胞膜染料及核仁素抗体的荧光共定位成像实验表明该探针能特异性靶向tumour细胞表面核仁素，使cDNA被竞争下来，Cy3荧光恢复，实现了特异性的靶向成像tumour细胞表面核仁素。更重要的是，该结构通过aptamer的亲和作用及cholesterol 的疏水作用锚定到细胞膜上，有效地抑制了细胞内吞作用，能长时间稳定存在于细胞表面。流式细胞术及荧光共聚焦成像结果表明，GC-chol-apt与细胞的亲和力提高了近100倍，并且能稳定锚定在细胞膜上长达6 小时，为长时间细胞膜成像提供了有力工具。

图：探针 GC-chol-apt-cDNA 用于细胞膜表面核仁素靶向成像。

结论：复合探针 GC-chol-apt-cDNA 能有效地靶向tumour细胞，诱导细胞Apoptosis 并抑制其增殖活性，其抑制率可高达81%左右。同时实现长效的膜蛋白成像，并通过诱导细胞表面核仁素交联，进而激活细胞内线粒体相关Apoptosis 信号，最终抑制体内外tumour细胞的增殖。