【摘要】：CD147為廣泛表達(dá)于人體各組織細(xì)胞的跨膜糖蛋白,通過與多種不同的配體結(jié)合,參與了各種生理、病理過程,如細(xì)胞運(yùn)動、組織重建、炎癥發(fā)生等。CD147在腫瘤細(xì)胞中呈異常高表達(dá),且能夠誘導(dǎo)鄰近成纖維細(xì)胞及腫瘤細(xì)胞自身產(chǎn)生胞外基質(zhì)金屬酶(MMPs),以降解胞外基質(zhì),促進(jìn)腫瘤新生血管的形成及癌細(xì)胞的浸潤轉(zhuǎn)移,因此,CD147又被稱為胞外基質(zhì)金屬酶誘導(dǎo)因子(EMMPRIN)。CD147這一功能的發(fā)揮與其糖基化狀態(tài)密切相關(guān),根據(jù)其糖鏈復(fù)雜程度及結(jié)構(gòu)不同,可以分為高糖基化(HG-CD147)和低糖基化(LG-CD147)兩種形式,功能研究表明,只有HG-CD147才能誘導(dǎo)MMPs的分泌,發(fā)揮促腫瘤轉(zhuǎn)移的功能。然而,目前針對CD147的檢測大都由其抗體發(fā)展而來,且僅能檢測CD147分子存在與否,而針對其糖基化修飾水平的檢測仍是一片空白。因此,在細(xì)胞及組織水平上,開展針對CD147功能實現(xiàn)的活性形式的檢測技術(shù)研究,對相關(guān)疾病及腫瘤轉(zhuǎn)移機(jī)制的深入研究具有重要意義。本工作研究并設(shè)計了靶向CD147糖基化修飾水平的基因編碼FRET熒光傳感器,并對其工作效率進(jìn)行了檢測。首先,我們采用與CD147特異結(jié)合的蛋白質(zhì)或多肽,作為傳感器中靶向CD147的識別結(jié)構(gòu),在此基礎(chǔ)上設(shè)計并合成了一系列針對CD147糖基化水平的熒光傳感器,并通過活細(xì)胞共定位實驗篩選得到靶向能力最好的一組傳感器;然后,我們通過熒光成像在活細(xì)胞水平上探究了該組傳感器對CD147糖基化修飾水平變化的響應(yīng),建立了熒光傳感器響應(yīng)與CD147糖基化水平變化的半定量關(guān)系;最后,我們又對熒光傳感器的CD147靶向特異性進(jìn)行了驗證。未來將在此系列熒光傳感器基礎(chǔ)上,進(jìn)一步完善其響應(yīng)效率,并有望利用熒光傳感器深入研究CD147糖基化與腫瘤轉(zhuǎn)移的關(guān)系機(jī)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.
【學(xué)位授予單位】：中國科學(xué)技術(shù)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q78;TP212

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