羧基對(duì)殼聚糖—硬脂酸嫁接物介導(dǎo)的基因傳遞的影響
發(fā)布時(shí)間：2018-11-24 14:51
【摘要】：本研究為了進(jìn)一步增加殼聚糖硬脂酸的轉(zhuǎn)染效率,分別從化學(xué)嫁接和物理混合兩種方式引入羧基,以研究促進(jìn)CSO-SA基因轉(zhuǎn)染效率的方法,并探討其機(jī)理。 通過(guò)碳二亞胺法,將小分子陰離子-順烏頭酸化學(xué)嫁接到CSO-SA上得到CA-CSO-SA。氫核磁共振確證了嫁接物的化學(xué)組成。經(jīng)TNBS法測(cè)得CSO-SA和CA-CSO-SA的實(shí)際氨基取代度為4.03%和6.31%,臨界膠束濃度80.3μg/L和78.4μg/L。微粒粒度與表面電位分析儀測(cè)得1.0mg/mL的CSO-SA和CA-CSO-SA膠束的數(shù)均粒徑為55.3±7.6nm和60.0±7.1nm,電位為38.3±2.8mV和30.8±1.0mV。透射電子顯微鏡結(jié)果顯示這兩種膠束呈不規(guī)則的球形,粒徑較小,分布均一。 分別制備了CSO-SA/DNA, CA-CSO-SA/DNA復(fù)合物,雖然在順烏頭酸修飾后,CA-CSO-SA與DNA密接略有下降但是依然可以有效的保護(hù)DNA免受酶降解的能力,且形成的復(fù)合物具有良好的血清穩(wěn)定性。CA-CSO-SA/DNA復(fù)合物在HEK293細(xì)胞介導(dǎo)的轉(zhuǎn)染效率達(dá)到37%,與CSO-SA/DNA相比,轉(zhuǎn)染效率增加一倍,優(yōu)于陽(yáng)性對(duì)照l(shuí)ipofectamineTM2000。雖然順烏頭酸修飾降低了細(xì)胞攝取的速率,但是在24h時(shí)細(xì)胞攝取總量是沒(méi)有差異的。CA-CSO-SA/DNA在HEK293細(xì)胞中,主要通過(guò)網(wǎng)格蛋白介導(dǎo)的內(nèi)吞作用,以及小窩蛋白介導(dǎo)的胞吞作用和巨胞飲作用。CSO-SA/DNA復(fù)合物容易陷在內(nèi)溶酶體中,但CA-CSO-SA/DNA能夠更有效的從內(nèi)溶酶體中逃逸出來(lái)并分布在細(xì)胞質(zhì)中。順烏頭酸修飾提高了CSO-SA的生物安全性,且在轉(zhuǎn)染劑量下對(duì)細(xì)胞基本不呈現(xiàn)毒性。 將聚谷氨酸PGA分別與DNA混合后,再與CSO-SA嫁接物膠束混合,制備不同N/P/C比CSO-SA/DNA/PGA三元復(fù)合物。隨著PGA增加,三元復(fù)合物起始粒徑變化不大,而在N/P/C比為10/1/6時(shí)粒徑顯著性增加,而多分散系數(shù)以及電位都隨著PGA的加入而出現(xiàn)明顯的下降趨勢(shì)。以綠色熒光蛋白質(zhì)粒(pEGFP-C1)為報(bào)告基因,流式細(xì)胞儀測(cè)定細(xì)胞轉(zhuǎn)染百分率的結(jié)果表明CSO-SA/DNA/PGA三元復(fù)合物的基因轉(zhuǎn)染效率隨著PGA先增加后降低；在N/P/C=10/1/4時(shí)轉(zhuǎn)染效率為27.4%,是CSO-SA/DNA二元復(fù)合物的兩倍,且接近陽(yáng)性LipofectamineTM2000對(duì)照,以蟲(chóng)熒光素酶質(zhì)粒為報(bào)告基因,化學(xué)發(fā)光儀測(cè)定其發(fā)光值得結(jié)果表明CSO-SA/DNA/PGA三元復(fù)合物在N/P/C=10/1/4蟲(chóng)熒光素酶的表達(dá)最高,達(dá)到5.32*105RLU/mg Protein,約是二元復(fù)合物表達(dá)的10倍。共聚焦觀察細(xì)胞內(nèi)過(guò)程和Bafilomycin Al抑制實(shí)驗(yàn)結(jié)果顯示CSO-SA/DNA/PGA復(fù)合物具有更高的溶酶體逃逸的效率歸結(jié)于三元復(fù)合物質(zhì)子緩沖能力的增加,從而獲得了更高效的基因轉(zhuǎn)染效率。 研究結(jié)果表明化學(xué)嫁接或者物理混合適量的負(fù)電荷都是有利于CSO-SA介導(dǎo)的基因轉(zhuǎn)染,且引入的羧基對(duì)于促進(jìn)內(nèi)溶酶體的逃逸起了重要作用。
[Abstract]:In order to further increase the transfection efficiency of chitosan stearic acid, carboxyl groups were introduced from two ways of chemical grafting and physical mixing in order to study the method of promoting the transfection efficiency of CSO-SA gene and its mechanism. Chemical grafting of small anions and cisaconitric acid onto CSO-SA to obtain CA-CSO-SA. by carbodiimide method The chemical composition of the graft was confirmed by HNMR. The actual degree of amino substitution for CSO-SA and CA-CSO-SA was 4.03% and 6.31, and the critical micelle concentration was 80.3 渭 g / L and 78.4 渭 g / L by TNBS method. The average particle size of CSO-SA and CA-CSO-SA micelles in 1.0mg/mL were 55.3 鹵7.6nm and 60.0 鹵7.1 nm, the potential were 38.3 鹵2.8mV and 30.8 鹵1.0 MV, respectively. The results of transmission electron microscope show that the two micelles are irregular spherical, smaller in size and uniform in distribution. The CSO-SA/DNA, CA-CSO-SA/DNA complexes were prepared separately. Although the close binding of CA-CSO-SA to DNA decreased slightly after modification of aconitate, it could still effectively protect DNA from enzymatic degradation. The transfection efficiency of CA-CSO-SA/DNA complex reached 37% in HEK293 cells. Compared with CSO-SA/DNA, the transfection efficiency of CA-CSO-SA/DNA complex was twice that of CSO-SA/DNA, which was better than that of positive control lipofectamineTM2000.. Although the rate of uptake was decreased by aconitonic acid modification, there was no difference in total uptake at 24 h. CA-CSO-SA/DNA in HEK293 cells mainly mediated endocytosis by grid protein. CSO-SA/DNA complex is easy to sink into the inner lysosome, but CA-CSO-SA/DNA can escape from the inner lysosome and distribute in the cytoplasm more effectively. The modification of aconitonic acid improved the biosafety of CSO-SA and showed no toxicity to the cells at the transfection dose. Poly (glutamate) PGA was mixed with DNA and then mixed with CSO-SA grafted micelle to prepare CSO-SA/DNA/PGA ternary complexes with different N/P/C ratios. With the increase of PGA, the initial particle size of the ternary complex does not change much, but the particle size increases significantly when the N/P/C ratio is 10-1-6, while the polydispersity coefficient and potential decrease obviously with the addition of PGA. Using green fluorescent protein (pEGFP-C1) as reporter gene, the transfection rate of CSO-SA/DNA/PGA ternary complex was determined by flow cytometry. The transfection efficiency of CSO-SA/DNA/PGA ternary complex increased firstly with PGA and then decreased. The transfection efficiency at N/P/C=10/1/4 was 27.4%, which was twice as high as that of the binary complex of CSO-SA/DNA, and was close to the positive LipofectamineTM2000 control. The plasmids of luciferase were used as reporter genes. The results of chemiluminescence assay showed that the expression of CSO-SA/DNA/PGA ternary complex in luciferase of N/P/C=10/1/4 worm was the highest, and the expression of 5.32*105RLU/mg Protein, was about 10 times as high as that of binary complex. The results of confocal observation of intracellular processes and Bafilomycin Al inhibition experiments showed that the higher efficiency of lysosomal escape of CSO-SA/DNA/PGA complex was attributed to the increase of proton buffer capacity of ternary complexes. Thus, a more efficient gene transfection efficiency was obtained. The results show that chemical grafting or physical mixing with proper amount of negative charge is beneficial to CSO-SA mediated gene transfection and the introduction of carboxyl groups plays an important role in promoting the escape of internal lysosomes.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R943
本文編號(hào)：2354137
[Abstract]:In order to further increase the transfection efficiency of chitosan stearic acid, carboxyl groups were introduced from two ways of chemical grafting and physical mixing in order to study the method of promoting the transfection efficiency of CSO-SA gene and its mechanism. Chemical grafting of small anions and cisaconitric acid onto CSO-SA to obtain CA-CSO-SA. by carbodiimide method The chemical composition of the graft was confirmed by HNMR. The actual degree of amino substitution for CSO-SA and CA-CSO-SA was 4.03% and 6.31, and the critical micelle concentration was 80.3 渭 g / L and 78.4 渭 g / L by TNBS method. The average particle size of CSO-SA and CA-CSO-SA micelles in 1.0mg/mL were 55.3 鹵7.6nm and 60.0 鹵7.1 nm, the potential were 38.3 鹵2.8mV and 30.8 鹵1.0 MV, respectively. The results of transmission electron microscope show that the two micelles are irregular spherical, smaller in size and uniform in distribution. The CSO-SA/DNA, CA-CSO-SA/DNA complexes were prepared separately. Although the close binding of CA-CSO-SA to DNA decreased slightly after modification of aconitate, it could still effectively protect DNA from enzymatic degradation. The transfection efficiency of CA-CSO-SA/DNA complex reached 37% in HEK293 cells. Compared with CSO-SA/DNA, the transfection efficiency of CA-CSO-SA/DNA complex was twice that of CSO-SA/DNA, which was better than that of positive control lipofectamineTM2000.. Although the rate of uptake was decreased by aconitonic acid modification, there was no difference in total uptake at 24 h. CA-CSO-SA/DNA in HEK293 cells mainly mediated endocytosis by grid protein. CSO-SA/DNA complex is easy to sink into the inner lysosome, but CA-CSO-SA/DNA can escape from the inner lysosome and distribute in the cytoplasm more effectively. The modification of aconitonic acid improved the biosafety of CSO-SA and showed no toxicity to the cells at the transfection dose. Poly (glutamate) PGA was mixed with DNA and then mixed with CSO-SA grafted micelle to prepare CSO-SA/DNA/PGA ternary complexes with different N/P/C ratios. With the increase of PGA, the initial particle size of the ternary complex does not change much, but the particle size increases significantly when the N/P/C ratio is 10-1-6, while the polydispersity coefficient and potential decrease obviously with the addition of PGA. Using green fluorescent protein (pEGFP-C1) as reporter gene, the transfection rate of CSO-SA/DNA/PGA ternary complex was determined by flow cytometry. The transfection efficiency of CSO-SA/DNA/PGA ternary complex increased firstly with PGA and then decreased. The transfection efficiency at N/P/C=10/1/4 was 27.4%, which was twice as high as that of the binary complex of CSO-SA/DNA, and was close to the positive LipofectamineTM2000 control. The plasmids of luciferase were used as reporter genes. The results of chemiluminescence assay showed that the expression of CSO-SA/DNA/PGA ternary complex in luciferase of N/P/C=10/1/4 worm was the highest, and the expression of 5.32*105RLU/mg Protein, was about 10 times as high as that of binary complex. The results of confocal observation of intracellular processes and Bafilomycin Al inhibition experiments showed that the higher efficiency of lysosomal escape of CSO-SA/DNA/PGA complex was attributed to the increase of proton buffer capacity of ternary complexes. Thus, a more efficient gene transfection efficiency was obtained. The results show that chemical grafting or physical mixing with proper amount of negative charge is beneficial to CSO-SA mediated gene transfection and the introduction of carboxyl groups plays an important role in promoting the escape of internal lysosomes.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R943
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