本文選題：熒光硅納米顆粒　切入點：基因載體　出處：《蘇州大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：隨著納米科學(xué)技術(shù)的飛速發(fā)展,各種各樣的納米材料被用于構(gòu)建非病毒基因載體。其中,具有熒光性能的基因載體因為能夠示蹤細(xì)胞內(nèi)基因運(yùn)輸?shù)南嚓P(guān)過程而倍受矚目。在基因運(yùn)輸?shù)倪^程中,長時程實時的示蹤成像可以讓人們更加綜合地理解基因載體的胞內(nèi)行為,進(jìn)而有利于更加合理地設(shè)計高性能的基因載體。近年來,零維熒光硅納米顆粒受到了人們的廣泛關(guān)注。因其具備優(yōu)越的光學(xué)性質(zhì)和良好的生物安全性,被認(rèn)為是生物成像應(yīng)用中潛在的理想熒光探針。在本文中,我們首先構(gòu)建了一種基于熒光硅納米顆粒的基因載體,并成功將其應(yīng)用于基因運(yùn)輸和熒光生物成像。在DNA的負(fù)載和保護(hù)試驗中,基于熒光硅納米顆粒的基因載體不僅具有相對較高的DNA負(fù)載率,而且能夠有效地保護(hù)DNA,避免DNA酶的降解。在基因轉(zhuǎn)染測試中,基于熒光硅納米顆粒的基因載體在人宮頸癌(HeLa)細(xì)胞的基因轉(zhuǎn)染效率約為35%,比相同條件下聚乙烯亞胺(Polyetherimide, PEI)的轉(zhuǎn)染效率要高�；跓晒夤杓{米顆粒的基因載體同時也具備良好的生物安全性,在與HeLa細(xì)胞共孵育培養(yǎng)時間長達(dá)24和48小時的時間內(nèi),均未產(chǎn)生明顯的細(xì)胞毒性,HeLa細(xì)胞仍然維持90%以上的存活率。其次,通過追蹤熒光硅納米顆粒強(qiáng)而穩(wěn)定的熒光,我們長時程實時地研究了基因載體的胞內(nèi)行為(比如：細(xì)胞內(nèi)吞、胞內(nèi)轉(zhuǎn)運(yùn)等),進(jìn)而揭示了基因載體與溶酶體的“復(fù)融合”(re-fusion)現(xiàn)象。我們選擇了另外一種典型的人乳腺癌(MCF-7)細(xì)胞作為對照組來研究載體的基因轉(zhuǎn)染和胞內(nèi)行為�；跓晒夤杓{米顆粒的載體在MCF-7細(xì)胞中的基因轉(zhuǎn)染效率約為16%,比相同實驗條件下HeLa細(xì)胞的轉(zhuǎn)染效率小的多。在活細(xì)胞長時程實時的示蹤成像中,MCF-7細(xì)胞中同樣存在基因載體與溶酶體的“復(fù)融合”現(xiàn)象。在相同的時間內(nèi)(15分鐘的實時示蹤成像),基因載體與溶酶體的“復(fù)融合”在MCF-7細(xì)胞中發(fā)生了3次,而在HeLa細(xì)胞中僅發(fā)生了1次。最后,通過進(jìn)一步分析此兩種細(xì)胞系的基因轉(zhuǎn)染效率和胞內(nèi)基因載體與溶酶體“復(fù)融合”現(xiàn)象之間的關(guān)系可知,“復(fù)融合”現(xiàn)象在基因轉(zhuǎn)染效率低的MCF-7細(xì)胞中比在基因轉(zhuǎn)染效率高的HeLa細(xì)胞中發(fā)生的次數(shù)更多。綜上可知,基于熒光硅納米顆粒的基因載體不僅具有強(qiáng)而穩(wěn)定的熒光和良好的生物安全性,而且具有相對較高的DNA負(fù)載和基因轉(zhuǎn)染能力。利用其優(yōu)異的光學(xué)性質(zhì),本文進(jìn)一步研究了基因載體的胞內(nèi)命運(yùn)。這些結(jié)果表明基于熒光硅納米顆粒的基因載體有望作為具有熒光性能的新型基因載體應(yīng)用于活細(xì)胞內(nèi)長時程實時的示蹤成像,并為人們提供更加有價值的信息來深入地了解和研究基因載體的胞內(nèi)行為。
[Abstract]:With the rapid development of nanotechnology, a variety of nanomaterials have been used to construct non-viral gene vectors. Gene vectors with fluorescence properties have attracted much attention because of their ability to trace the related processes of gene transport within cells. In the process of gene transport, long-term real-time tracer imaging can provide a more comprehensive understanding of the intracellular behavior of gene vectors. In recent years, zero-dimensional fluorescent silicon nanoparticles have attracted wide attention because of their superior optical properties and good biosafety. In this paper, we first constructed a gene vector based on fluorescent silicon nanoparticles. In the loading and protection experiments of DNA, the gene carrier based on fluorescent silicon nanoparticles not only has a relatively high DNA loading rate, but also has been successfully applied to gene transport and fluorescence imaging. And it can effectively protect the DNA from the degradation of DNA enzyme. The transfection efficiency of the gene vector based on fluorescent silicon nanoparticles in human cervical cancer cell line HeLa was about 35, which was higher than that of PEI under the same conditions. The gene vector based on fluorescent silicon nanoparticles was also found to be more efficient than that of PEI under the same conditions. With good biological safety, After incubation with HeLa cells for 24 and 48 hours, no significant cytotoxicity was observed. The survival rate of HeLa cells remained above 90%. Secondly, the strong and stable fluorescence of fluorescent silicon nanoparticles was traced. We have studied the intracellular behavior of gene vectors (E. G. endocytosis) over a long period of time. We selected another typical human breast cancer cell line MCF-7 as control group to study the gene transfection and intracellular behavior of the vector. The transfection efficiency of fluorescent silicon nanoparticles in MCF-7 cells was about 16, which was much lower than that of HeLa cells under the same experimental conditions. The gene was also present in MCF-7 cells in live cell long time and real time tracer imaging. The phenomenon of "refusion" of vector and lysosome. In the same time, the "fusion" of gene vector and lysosome took place three times in MCF-7 cells after 15 minutes of real-time tracer imaging. But only once in HeLa cells. Finally, Through further analysis of the gene transfection efficiency of the two cell lines and the relationship between the intracellular gene vector and lysosomal "refusion" phenomenon, it can be seen that the "refusion" phenomenon in MCF-7 cells with low gene transfection efficiency is more than that in the gene transfer cell line. HeLa cells with high dyeing efficiency occur more frequently. The gene vector based on fluorescent silicon nanoparticles not only has strong and stable fluorescence and good biological safety, but also has relatively high DNA loading and gene transfection ability. In this paper, the intracellular fate of gene vectors has been further studied. These results indicate that the novel gene vectors based on fluorescent silicon nanoparticles are expected to be used as novel gene vectors with fluorescent properties for real-time tracer imaging in living cells for a long time. And provide more valuable information for people to understand and study the intracellular behavior of gene vectors.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：Q78

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