本文選題：分子傘 + 藥物載體　； 參考：《廈門大學》2008年碩士論文

【摘要】： 細胞膜作為細胞的一道天然屏障,使得許多具有治療作用的藥物和大分子生物活性物質,特別是高親水性和帶電物質,如反義寡聚核苷酸、DNA、蛋白質和多肽等難以通過細胞膜。構建一種藥物載體使其能順利穿透細胞膜并在靶向部位釋放,已成為藥物設計中亟需解決的問題之一。一種具有雙親性的分子傘載體有望攜帶上述藥物實現跨膜運輸,并解決許多載體在治療過程中存在的穿膜效率低和安全性等問題。本論文的研究內容主要包括設計合成帶有綠色熒光的5-羧基熒光素分子傘,考察其穿透細胞膜的能力和體外細胞毒性,探討分子傘載體可能的穿膜機制。研究結果如下: (一)分子傘的合成與表征 (1)在無水有機溶劑中,膽酸分子與N-羥基琥珀酰亞胺(NHS)在縮合劑N,N′-二環(huán)己基碳二亞胺(DCC)的作用下反應得到膽酸-NHS活化酯,然后以二異丙基乙胺(DIPEA)為縛酸堿,在無水二甲基甲酰胺中膽酸-NHS活化酯與亞精胺的兩個端氨基偶聯(lián),形成含有一個活性氨基(仲胺)的雙臂分子傘,即N_1,N_3-亞精胺二膽酸酰胺。 (2)在無水二甲基甲酰胺中,5-羧基熒光素與3-羥基-1,2,3-苯并三嗪-4(3H)-酮(Dhbt)在縮合劑N,N′-二環(huán)己基碳二亞胺(DCC)的作用下活化得到5-羧基熒光素的Dhbt活化酯,再以三乙胺為縛酸堿在無水二甲基甲酰胺中與上述雙臂分子傘的活性氨基偶聯(lián),形成5-羧基熒光素分子傘(5-cf-mu)。 (3)對各步產物如膽酸-NHS活化酯、N_1,N_3-亞精胺二膽酸酰胺和5-cf-mu的結構和分子量分別采用紅外光譜法、核磁共振譜法和質譜法進行確定,同時對這三步產物的熔點進行測定;5-cf-mu的純度采用高效液相色譜法進行分析。結果表明:這三步產物的各種表征結果與其相應分子結構、分子量都完全符合,5-cf-mu的純度在95%以上。 (二)分子傘的穿膜性能研究 (1)MTT法檢測了濃度分別為25,50,100和200μM的5-cf-mu在24和48h內對L929細胞的細胞毒性,結果表明:24 h內上述濃度的5-cf-mu對L929細胞均沒有毒性;25μM的5-cf-mu在48 h內也沒有毒性,50,100和200μM在48 h呈現少許毒性,但仍有較高的細胞成活率。同時檢測了5-cf-mu對Hela細胞在24 h內的細胞毒性,結果表明濃度低于100μM時,分子傘對Hela細胞也沒有毒性。上述結果說明分子傘具有良好的生物相容性。 (2)采用熒光顯微鏡和激光共聚焦顯微鏡檢測了5-cf-mu在Hela細胞和L929細胞內的分布情況。綠色熒光點不僅分布在細胞質中,而且分布在細胞核周圍,表明5-cf-mu已經進入了Hela細胞和L929細胞。實驗證明分子傘是一種可以實現跨膜運輸的載體。 (3)采用流式細胞儀考察了溫度、濃度、時間以及ATP抑制劑作用下對5-cf-mu穿透細胞膜的效率的影響。結果表明分子傘的穿膜是一個時間、濃度和溫度依賴而非能量依賴的過程。 (三)分子傘的穿膜機制 (1)建立了分子傘穿透細胞膜的數學模型。根據流式細胞儀檢測結果,細胞內的平均熒光強度(MFI)與培養(yǎng)時間的平方根(t~(1/2))、初始濃度的平方根(C_o~(1/2))均成很好的線性關系,線性相關系數(R值)分別為0.985和0.994。 (2)分子傘穿透細胞膜的過程符合所建立的數學模型,表明分子傘是以溶解—擴散的方式穿透細胞膜。
[Abstract]:The invention relates to a molecular umbrella carrier with amphipathy , which is expected to carry the drug to realize transmembrane transport and solve the problems of low membrane penetrating efficiency and safety existing in the drug design .



( 1 ) Synthesis and Characterization of Molecular umbrella



( 1 ) In an anhydrous organic solvent , the cholic acid molecule reacts with N - hydroxysuccinimide ( NHS ) under the action of a condensing agent N , N ' - dicyclohexylcarbodiimide ( DCC ) to obtain the cholic acid - NHS activated ester , and then the cholic acid - NHS activated ester is coupled with the two end - amino groups of the spermidine in the anhydrous dimethylformamide to form a double - arm molecular umbrella containing an active amino group ( secondary amine ) , namely , N _ 1 , N _ 3 - spermidine di - cholic acid amide .



( 2 ) 5 - carboxyfluorescein and 3 - hydroxy - 1 , 2 , 3 - benzotriazine - 4 ( 3H ) - one ( dhbt ) are activated under the action of condensing agent N , N ' - dicyclohexylcarbodiimide ( DCC ) under the action of N , N ' - dicyclohexylcarbodiimide ( DCC ) under the action of condensing agent N , N ' - dicyclohexylcarbodiimide ( DCC ) , and then triethylamine is used as acid - binding base to be coupled with the active amino group of the double - arm molecular umbrella in anhydrous dimethylformamide to form a 5 - carboxyfluorescein molecular umbrella ( 5 - cf - mu ) .



( 3 ) The structure and molecular weight of the products such as cholic acid - NHS activated ester , N _ 1 , N _ 3 - spermidine - dicholic acid amide and 5 - cf - mu were determined by infrared spectroscopy , nuclear magnetic resonance spectroscopy and mass spectrometry , respectively . The purity of these three - step products was determined by high performance liquid chromatography . The results showed that all the characterization results of these three products were consistent with their molecular structure and molecular weight , and the purity of 5 - cf - mu was more than 95 % .



Study on the Film Properties of ( II ) Molecular Umbrella



( 1 ) The cytotoxicity of 5 - cf - mu of 25 , 50 , 100 and 200 渭M on L929 cells was detected by MTT assay . The results showed that 5 - cf - mu of 25 渭M had no toxicity to L929 cells .



( 2 ) The distribution of 5 - cf - mu in Hela cells and L929 cells was detected by a fluorescence microscope and a laser confocal microscope . The green fluorescent dots were not only distributed in the cytoplasm but also around the nucleus , indicating that 5 - cf - mu had entered Hela cells and L929 cells .



( 3 ) The effect of temperature , concentration , time and ATP inhibitor on the efficiency of 5 - cf - mu penetrate cell membrane was investigated by flow cytometry . The results showed that the membrane of molecular umbrella was a time , concentration and temperature dependence rather than energy dependence .



( 3 ) Film - penetrating mechanism of molecular umbrella



According to the results of flow cytometry , the mean fluorescence intensity ( MFI ) and the square root of the culture time ( t ~ ( 1 / 2 )) , the square root of the initial concentration ( C _ o ~ ( 1 / 2 )) were linear , and the linear correlation coefficient ( R value ) was 0.985 and 0.994 , respectively .



( 2 ) The process of penetrating the cell membrane by the molecular umbrella accords with the established mathematical model , indicating that the molecular umbrella penetrates the cell membrane in a dissolved - diffusion way .
【學位授予單位】：廈門大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R341

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本文編號：1947362