本文選題：CD22 + 靶向識(shí)別��； 參考：《大連醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:基于受體-配體(receptor-ligand)間相互特異性識(shí)別原理,借助化學(xué)生物學(xué)偶聯(lián)的手段,設(shè)計(jì)、合成具有CD22靶向功能的熒光納米材料,表征熒光納米材料的材料性質(zhì),并通過細(xì)胞靶向?qū)嶒?yàn)實(shí)現(xiàn)CD22高表達(dá)的細(xì)胞系的示蹤,為CD22的生物學(xué)診斷提供有力工具。方法:1.基于receptor-ligand相互識(shí)別原理,借助N-乙酰神經(jīng)氨酸(NANA)對(duì)CD22的識(shí)別作用,對(duì)其進(jìn)行衍生化處理,得到不穩(wěn)定的O-�；惢�(NANA-EDC),用于氨基量子點(diǎn)(NH2-QDs)的生物偶聯(lián);將NANA-EDC與NH2-QDs進(jìn)行偶聯(lián),得到NANA-QDs納米復(fù)合物。2.對(duì)NANA-QDs納米復(fù)合物的材料性質(zhì)進(jìn)行表征,通過測(cè)定NANA-QDs納米復(fù)合物中配體NANA的偶聯(lián)度,粒徑分布,Zeta電位,量子產(chǎn)率,光譜學(xué)性質(zhì),生物毒性等性質(zhì),明確NANA-QDs納米復(fù)合物的結(jié)構(gòu),熒光性能和安全性。3.借助體外細(xì)胞實(shí)驗(yàn)對(duì)NANA-QDs納米復(fù)合物的靶向性能進(jìn)行評(píng)價(jià),通過模式CD22高表達(dá)細(xì)胞株Daudi表面熒光分布、競(jìng)爭(zhēng)性結(jié)合實(shí)驗(yàn)、納米粒子細(xì)胞內(nèi)吞實(shí)驗(yàn)、免疫熒光共定位等實(shí)驗(yàn)確定NANA-QDs特異性和親和性。4.將NANA-QDs納米復(fù)合物用于多種腫瘤細(xì)胞的CD22表達(dá)水平檢測(cè),并通過傳統(tǒng)方法蛋白質(zhì)免疫印跡(Western blotting analysis)、流式細(xì)胞術(shù)、細(xì)胞免疫熒光對(duì)結(jié)果可靠性進(jìn)行驗(yàn)證。結(jié)果:經(jīng)TEM及DLS測(cè)試,結(jié)果表明NANA-QDs納米復(fù)合物的平均粒徑分布為10nm左右且分布均勻;平均偶聯(lián)度為1分子QD納米粒子表面可偶聯(lián)294個(gè)NANA分子,配體分子濃度較高,有利于下一步細(xì)胞靶向?qū)嶒?yàn);偶聯(lián)后,量子產(chǎn)率穩(wěn)定,保持在70%左右,且熒光光譜譜峰對(duì)稱,熒光性能良好;在細(xì)胞靶向性測(cè)試中,NANA-QDs納米粒子在Daudi細(xì)胞表面產(chǎn)生強(qiáng)烈的紅色熒光信號(hào),初期熒光信號(hào)具有類似細(xì)胞膜結(jié)構(gòu)分布,而陰性對(duì)照中熒光微弱且無特異性分布;通過與游離NANA的競(jìng)爭(zhēng)性結(jié)合及免疫熒光共定位實(shí)驗(yàn)表明,NANA-QDs納米復(fù)合物與Daudi細(xì)胞的結(jié)合是NANA上環(huán)狀結(jié)構(gòu)對(duì)CD22的特異性識(shí)別產(chǎn)生;納米粒子細(xì)胞動(dòng)力學(xué)實(shí)驗(yàn)表明,隨著時(shí)間延長(zhǎng),熒光分布逐漸由Daudi細(xì)胞表面進(jìn)入細(xì)胞內(nèi)部,表明NANA-QDs可通過細(xì)胞的CD22內(nèi)吞作用進(jìn)入細(xì)胞;以上一系列實(shí)驗(yàn)表明NANA-QDs對(duì)細(xì)胞表面CD22具有特異性識(shí)別及靶向作用。將NANA-QDs納米粒子用于淋巴瘤、肝癌、肺癌及乳腺癌六種腫瘤細(xì)胞CD22表達(dá)水平的測(cè)定,表明CD22除在淋巴瘤細(xì)胞特異性高表達(dá)外,在肝癌細(xì)胞、肺癌及乳腺癌細(xì)胞均呈低表達(dá),在A549細(xì)胞中CD22的表達(dá)隨傳代次數(shù)的增加呈現(xiàn)上調(diào)趨勢(shì);利用細(xì)胞免疫熒光、流式細(xì)胞術(shù)和Westerning blotting等傳統(tǒng)鑒定手段驗(yàn)證了上述實(shí)驗(yàn)結(jié)果的可靠性。結(jié)論:本研究利用NANA環(huán)狀結(jié)構(gòu)特異性識(shí)別CD22受體的原理,制備了NANA-QDs納米熒光材料,其粒徑分布均勻,熒光性能良好,低生物毒性,對(duì)細(xì)胞表面跨膜糖蛋白CD22具有較強(qiáng)的靶向性,為CD22的生物學(xué)診斷提供了有力工具。
[Abstract]:Aim: to design and synthesize fluorescent nanomaterials with CD22 targeting function based on the principle of mutual specificity recognition between receptor and ligand (receptor-ligand), and to characterize the properties of fluorescent nanomaterials. The high expression of CD22 cell line can be traced by cell targeting experiment, which provides a powerful tool for the biological diagnosis of CD22. Method 1: 1. Based on the principle of receptor-ligand mutual recognition, the unsteady O-acyl isoester (NA-EDC) was obtained by using the recognition of N-acetylneuraminic acid (NANA-EDC) on CD22, which was used to couple amino quantum dots (NH2-QDs), and NANA-EDC was coupled with NH2-QDs. NANA-QDs nanocomposites. The properties of NANA-QDs nanocomposites were characterized. The structure of NANA-QDs nanocomposites was determined by determining the coupling degree, particle size distribution, quantum yield, spectroscopic properties and biotoxicity of NANA-QDs nanocomposites. Fluorescence properties and safety. The targeting properties of NA-QDs nanocomposites were evaluated by cell experiments in vitro. The surface fluorescence distribution, competitive binding assay and endocytosis of NA-QDs nanocomposites were determined by the model CD22 high expression cell line Daudi. The specificity and affinity of NANA-QDs were determined by immunofluorescence co-localization. NANA-QDs nanocomplexes were used to detect the expression of CD22 in various tumor cells, and the reliability of the results was verified by Western blotting analysis), flow cytometry (FCM). Results: the results of TEM and DLS showed that the average particle size distribution of NANA-QDs nanocomposites was about 10nm and the average coupling degree was 1 molecular QD nanoparticles. After coupling, the quantum yield is stable, about 70%, and the fluorescence spectrum peak is symmetrical, and the fluorescence performance is good. In the cell targeting test, NANA-QDs nanoparticles produced strong red fluorescence signals on the surface of Daudi cells. The initial fluorescence signals were similar to the cell membrane structure distribution, but the fluorescence in negative controls was weak and non-specific. The results of competitive binding with NANA and immunofluorescence co-localization showed that the binding of NANA-QDs nanoparticles to Daudi cells was due to the specific recognition of CD22 by the ring structure on NANA. The fluorescence distribution gradually entered the cells from the surface of Daudi cells, indicating that NANA-QDs could enter the cells through the endocytosis of CD22, and the above series of experiments showed that NA-QDs had a specific recognition and targeting effect on the CD22 on the cell surface. NANA-QDs nanoparticles were used to detect the expression of CD22 in lymphoma, liver cancer, lung cancer and breast cancer cells. The results showed that CD22 expression was low in hepatoma cells, lung cancer cells and breast cancer cells, except for the specific high expression of CD22 in lymphoma cells. The expression of CD22 in A549 cells was up-regulated with the increase of passage times, and the reliability of the above results was verified by traditional identification methods such as cellular immunofluorescence, flow cytometry and Westerning blotting. Conclusion: in this study, NANA-QDs nanofluorescent materials were prepared by the principle of NANA ring structure specific recognition of CD22 receptor. The NANA-QDs nanophore materials have the advantages of uniform particle size distribution, good fluorescence performance, low biotoxicity and strong targeting to transmembrane glycoprotein CD22 on cell surface. It provides a powerful tool for the biological diagnosis of CD 22.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：TB383.1;R73

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