CD147糖基化水平检测的基因编码FRET荧光传感器的设计、表达及应用

发布时间：2018-07-24 21:36

【摘要】：CD147为广泛表达于人体各组织细胞的跨膜糖蛋白,通过与多种不同的配体结合,参与了各种生理、病理过程,如细胞运动、组织重建、炎症发生等。CD147在肿瘤细胞中呈异常高表达,且能够诱导邻近成纤维细胞及肿瘤细胞自身产生胞外基质金属酶(MMPs),以降解胞外基质,促进肿瘤新生血管的形成及癌细胞的浸润转移,因此,CD147又被称为胞外基质金属酶诱导因子(EMMPRIN)。CD147这一功能的发挥与其糖基化状态密切相关,根据其糖链复杂程度及结构不同,可以分为高糖基化(HG-CD147)和低糖基化(LG-CD147)两种形式,功能研究表明,只有HG-CD147才能诱导MMPs的分泌,发挥促肿瘤转移的功能。然而,目前针对CD147的检测大都由其抗体发展而来,且仅能检测CD147分子存在与否,而针对其糖基化修饰水平的检测仍是一片空白。因此,在细胞及组织水平上,开展针对CD147功能实现的活性形式的检测技术研究,对相关疾病及肿瘤转移机制的深入研究具有重要意义。本工作研究并设计了靶向CD147糖基化修饰水平的基因编码FRET荧光传感器,并对其工作效率进行了检测。首先,我们采用与CD147特异结合的蛋白质或多肽,作为传感器中靶向CD147的识别结构,在此基础上设计并合成了一系列针对CD147糖基化水平的荧光传感器,并通过活细胞共定位实验筛选得到靶向能力最好的一组传感器;然后,我们通过荧光成像在活细胞水平上探究了该组传感器对CD147糖基化修饰水平变化的响应,建立了荧光传感器响应与CD147糖基化水平变化的半定量关系;最后,我们又对荧光传感器的CD147靶向特异性进行了验证。未来将在此系列荧光传感器基础上,进一步完善其响应效率,并有望利用荧光传感器深入研究CD147糖基化与肿瘤转移的关系机制。
[Abstract]:CD147 is a transmembrane glycoprotein widely expressed in human tissues and cells. By binding with many different ligands, CD147 participates in various physiological and pathological processes, such as cell movement, tissue reconstruction, inflammation, and so on. CD147 is highly expressed in tumor cells. In addition, extracellular matrix metalloproteinase (MMPs),) can be induced by the adjacent fibroblasts and tumor cells to degrade extracellular matrix, promote tumor angiogenesis and tumor cell invasion and metastasis. Therefore, the function of CD147, also known as extracellular matrix metallozyme inducible factor (EMMPRIN). CD147), is closely related to its glycosylation state. According to the complexity and structure of its sugar chain, it can be divided into two forms: high glycosylation (HG-CD147) and low glycosylation (LG-CD147). Functional studies show that only HG-CD147 can induce the secretion of MMPs and play a role in tumor metastasis. However, at present, the detection of CD147 is mostly developed by its antibody, and can only detect the presence or not of CD147 molecules, but the detection of glycosylation level of CD147 is still a blank. Therefore, it is of great significance to study the activity forms of CD147 at the cellular and tissue level, which is of great significance for the further study of related diseases and the mechanism of tumor metastasis. In this work, a novel FRET fluorescence sensor targeting CD147 glycosylation level was designed and its efficiency was tested. Firstly, we designed and synthesized a series of fluorescent sensors aimed at the glycosylation level of CD147 by using proteins or polypeptides specifically bound to CD147 as the recognition structure of targeted CD147 in the sensor. A group of sensors with the best targeting ability was obtained by the living cell co-localization experiment. Then, we studied the response of the sensors to the change of CD147 glycosylation level at the living cell level by fluorescence imaging. The semi-quantitative relationship between the response of the fluorescence sensor and the level of glycosylation of CD147 was established. Finally, the specificity of the CD147 target of the fluorescence sensor was verified. On the basis of this series of fluorescent sensors, its response efficiency will be further improved in the future, and the mechanism of CD147 glycosylation and tumor metastasis will be further studied by fluorescence sensors.
【学位授予单位】：中国科学技术大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78;TP212

【相似文献】