本文關鍵詞： 磷酸鈣納米顆粒 蛋白質載體 GFP KillerRed　出處：《中國科學技術大學》2015年碩士論文　論文類型：學位論文

【摘要】：蛋白質能催化生化反應,組成受體和細胞膜通道,提供細胞骨架支持,在細胞內轉運分子,是體內功能最多樣化的生物大分子。蛋白質藥物廣泛的應用于癌癥治療、疫苗、再生藥物、治療基因缺陷疾病和成像等領域。然而,由于蛋白質特有的性質,比如較大的體積、不同的表面電勢、易受損傷的三級結構,將目標蛋白運載入細胞內有很多阻礙。所以蛋白質運載策略中最大的挑戰(zhàn)是在不破壞蛋白質結構和生物活性的前提下將蛋白運載到細胞質。大多數蛋白質運載體系依賴于細胞的內吞作用。因此,被運載的目標蛋白很容易在內體／溶酶體弱酸性的采環(huán)境中被降解,進而無法到達目標的亞細胞區(qū)域發(fā)揮作用�；谝陨显�,我們用了一種pH敏感的納米生物材料,來避免目標蛋白在內體／溶酶體中降解,并有效的釋放到細胞質中。磷酸鈣納米顆粒是pH敏感的蛋白質運載體系,有很好的生物相容性和生物降解性。在本論文中,我們以磷酸鈣納米顆粒為基礎建立了一種有效的細胞內蛋白質運載體系。我們設計了一種簡單低成本的磷酸鈣納米顆粒制備方法,檢測了納米顆粒各方面的性質,包括顆粒粒徑、顆粒形態(tài)、載藥率、包封率、pH敏感性質和穩(wěn)定性。磷酸鈣納米顆粒有很好的分散性并保持穩(wěn)定的粒徑。在內體／溶酶體的弱酸性環(huán)境中,蛋白能很快從顆粒上釋放,而在生理環(huán)境(pH=7.4)中能保持穩(wěn)定。然后,我們進一步研究了磷酸鈣納米顆粒的細胞攝取、蛋白在細胞內的釋放情況以及顆粒上負載蛋白的生物活性。一方面,我們通過熒光顯微鏡和流式細胞術來研究磷酸鈣納米顆粒的細胞攝取情況；結果表明顆粒能成功有效的被細胞攝取。由于內體／溶酶體逃逸對于細胞內藥物的釋放是一個阻礙,我們通過激光共聚焦顯微鏡觀察蛋白在細胞內的釋放情況：結果表明蛋白質能成功釋放到細胞質中。另一方面,SDS-PAGE實驗和圓二色譜分析結果表明由于溫和的制備方法,磷酸鈣納米顆粒上釋放的蛋白能保持其結構不被破壞。另外,磷酸鈣納米顆粒上釋放的KillerRed蛋白保留了其生物活性。以上的實驗結果表明磷酸鈣納米顆粒是一種合適的蛋白質載體。
[Abstract]:Proteins catalyze biochemical reactions, form receptors and cell membrane channels, provide cytoskeleton support, transport molecules within cells, and are the most versatile biological macromolecules in the body. Regenerative drugs, treatment of genetic defects, imaging, etc. However, because of the specific properties of proteins, such as large volume, different surface potential, vulnerable tertiary structure, There are many obstacles to carrying the target protein into the cell. So the biggest challenge in the protein delivery strategy is to transport the protein to the cytoplasm without destroying the protein structure and biological activity. Most protein carriers. The lineage depends on the endocytosis of the cell. The target protein is easily degraded in a weakly acidic inner / lysosomal environment, which can't reach the target subcellular area. For these reasons, we use a pH sensitive nanomaterials. In order to avoid degradation of target protein in inner body / lysosome and release it into cytoplasm effectively. Calcium phosphate nanoparticles are a pH sensitive protein delivery system with good biocompatibility and biodegradability. We have established an effective intracellular protein delivery system based on calcium phosphate nanoparticles. We have designed a simple and low-cost method for preparing calcium phosphate nanoparticles and examined their properties. Including particle size, particle morphology, drug loading rate, entrapment efficiency, pH sensitivity and stability. Calcium phosphate nanoparticles have good dispersion and maintain stable particle size. The protein was released quickly from the particles and remained stable in the physiological environment (pH = 7.4). Then we further studied the cellular uptake of calcium phosphate nanoparticles. On the one hand, we studied the uptake of calcium phosphate nanoparticles by fluorescence microscopy and flow cytometry. The results showed that the granules were successfully and effectively ingested by the cells, because the escape of the inner body / lysosome was an obstacle to the release of the drugs in the cell. We observed the release of proteins in cells by laser confocal microscopy: the results showed that proteins could be successfully released into the cytoplasm. On the other hand, SDS-PAGE and circular dichroism analysis showed that due to mild preparation methods, The protein released on the calcium phosphate nanoparticles can keep its structure intact. The KillerRed protein released on the calcium phosphate nanoparticles retains its biological activity. The above results show that the calcium phosphate nanoparticles are a suitable protein carrier.
【學位授予單位】：中國科學技術大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：O614.231;TB383.1

【共引文獻】

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