本文關(guān)鍵詞：α-螺旋型膜活性多肽對(duì)模擬膜和生物膜作用機(jī)理的研究　出處：《吉林大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 膜活性多肽 模擬膜 細(xì)胞膜 選擇性 原子力顯微鏡 單分子力譜技術(shù)

【摘要】：在臨床治療中,細(xì)菌感染一直是備受人們關(guān)注的問題�？股氐某霈F(xiàn)有效抑制各種致病細(xì)菌、真菌的感染,對(duì)保護(hù)人類的健康起著重要的作用。但是,抗生素的濫用導(dǎo)致耐藥菌甚至是超級(jí)細(xì)菌的產(chǎn)生,為人類帶來(lái)新的問題。因此,尋找抗生素的合適替代品成為人們研究的熱點(diǎn)。同時(shí),癌癥作為一種常見的惡性疾病,發(fā)病率呈現(xiàn)逐年升高的趨勢(shì),嚴(yán)重威脅著人類的健康和生命。目前臨床使用的抗腫瘤藥物主要是烷基化試劑和抗代謝類藥物,很多藥物不僅利用率低,而且具有很強(qiáng)的毒副作用。因此,尋找新型的抗腫瘤藥物也具有十分重要的意義。膜活性多肽是一類作用于細(xì)胞膜的生物活性肽,主要包括抗細(xì)菌肽、抗真菌肽和抗癌肽。膜活性多肽以其廣譜性、速效性和不易產(chǎn)生耐藥性等特點(diǎn)而受到人們的廣泛重視。但是,膜活性多肽對(duì)細(xì)菌和癌細(xì)胞作用機(jī)理尚無(wú)定論。因此,研究膜活性多肽的作用機(jī)理具有重要的理論和實(shí)際意義。本課題以前期工作獲得的膜活性多肽V13K為母肽,利用氨基酸之間疏水性和電荷不同的性質(zhì),改造設(shè)計(jì)兩組具有不同疏水性和電荷性質(zhì)的多肽。首先,通過(guò)采用倍比稀釋法檢測(cè)多肽對(duì)不同的革蘭氏陽(yáng)性細(xì)菌和陰性細(xì)菌的抗細(xì)菌活性、針對(duì)不同的癌細(xì)胞的抗癌活性以及針對(duì)正常細(xì)胞和血紅細(xì)胞的毒性,通過(guò)構(gòu)效關(guān)系分析得到膜活性多肽的生物活性與多肽的疏水性和電荷密切相關(guān),疏水性越強(qiáng),其抗細(xì)菌和抗癌的能力越強(qiáng);含有負(fù)電荷氨基酸的多肽生物活性相對(duì)較弱。同時(shí),實(shí)驗(yàn)證實(shí)膜活性多肽對(duì)細(xì)菌和癌細(xì)胞的破壞能力要高于對(duì)正常細(xì)胞和血紅細(xì)胞的破壞能力。其次,通過(guò)膜活性多肽對(duì)細(xì)菌外膜和內(nèi)膜滲透性試驗(yàn),和流式細(xì)胞儀檢測(cè)其對(duì)癌細(xì)胞膜的破壞作用。結(jié)果證實(shí),膜活性多肽對(duì)細(xì)菌和癌細(xì)胞的細(xì)胞膜均具有破壞能力。并且,疏水性越強(qiáng)的多肽對(duì)細(xì)菌和癌細(xì)胞的細(xì)胞膜破壞能力越強(qiáng)。此外,含有負(fù)電荷氨基酸的膜活性多肽對(duì)細(xì)菌和癌細(xì)胞的細(xì)胞膜破壞能力相對(duì)較弱,這一結(jié)果與膜活性多肽的生物活性實(shí)驗(yàn)結(jié)果相一致。進(jìn)一步利用磷脂模擬膜方法,通過(guò)色氨酸藍(lán)移實(shí)驗(yàn)和碘化鉀淬滅實(shí)驗(yàn)研究膜活性多肽與細(xì)胞膜磷脂成分相互作用的過(guò)程。結(jié)果證實(shí),疏水性強(qiáng)的多肽與磷脂模擬膜的作用能力強(qiáng);含有負(fù)電荷氨基酸的多肽與磷脂模擬膜的作用能力較弱。此外,我們還發(fā)現(xiàn)含有正電荷的膜活性多肽與含有負(fù)電荷的原核細(xì)胞模擬膜和癌細(xì)胞模擬膜的作用能力相對(duì)較強(qiáng),而多肽與中性的正常細(xì)胞模擬膜的作用能力相對(duì)較弱。這一結(jié)果與膜活性多肽的生物活性實(shí)驗(yàn)結(jié)果相一致,進(jìn)一步證明膜活性多肽對(duì)細(xì)菌細(xì)胞膜和癌細(xì)胞膜具有選擇性。最后,通過(guò)原子力顯微鏡成像技術(shù)和單分子力譜技術(shù),進(jìn)一步研究膜活性多肽對(duì)不同細(xì)胞膜作用的機(jī)理。其中,原子力顯微鏡成像技術(shù)可以直觀的展現(xiàn)膜活性多肽與細(xì)菌和癌細(xì)胞相互作用前后的細(xì)胞膜表面形貌。原子力顯微鏡單分子力譜技術(shù)研究單分子多肽與不同磷脂模擬膜及其對(duì)應(yīng)的生物膜的相互作用力大小。通過(guò)對(duì)比分析膜活性多肽對(duì)不同細(xì)胞膜作用的力值大小和作用幾率,證實(shí)疏水性越強(qiáng)的多肽與細(xì)胞膜作用的能力越強(qiáng),而含有負(fù)電荷氨基酸的多肽作用能力較弱。此外,利用膜活性多肽與原核細(xì)胞膜和癌細(xì)胞膜的作用能力較強(qiáng),對(duì)正常細(xì)胞膜作用能力相對(duì)較弱。上述結(jié)果進(jìn)一步驗(yàn)證膜活性多肽的靶向性作用機(jī)制主要依賴于細(xì)胞膜磷脂成分的差異,即含有正電荷的多肽與含有負(fù)電荷的原核細(xì)胞和癌細(xì)胞的細(xì)胞膜作用能力更強(qiáng),這一結(jié)果與前期提出的“膜區(qū)分機(jī)理”相一致。膜活性多肽對(duì)不同細(xì)胞膜作用的靶向特異性,為進(jìn)一步改造設(shè)計(jì)針對(duì)細(xì)菌和癌細(xì)胞靶向治療的膜活性多肽及其臨床應(yīng)用奠定重要的理論基礎(chǔ)。
[Abstract]:In the clinical treatment of bacterial infections has always been a concern of the people. The emergence of antibiotic effectively suppress various pathogenic bacteria, fungal infection, plays an important role in the protection of human health. However, the abuse of antibiotic resistant bacteria and even lead to the birth of super bacteria, bring new problems for human beings. Therefore, looking for a suitable alternative to antibiotics has become a hot research spot. At the same time, cancer as a common malignant disease, the incidence increasing year by year, a serious threat to human health and life. The current clinical use of anticancer drugs is an alkylating agent and anti metabolic drugs, many drug not only low utilization rate, but also has strong side effects. Therefore, it is very important to find new anticancer drug. The membrane is a kind of bioactive peptides on the cell membrane of biological active peptide, the main To include antibacterial peptide, antifungal peptides and anticancer peptides. Membrane active peptides with broad-spectrum, quick and easy to produce drug resistance and other characteristics have attracted much attention. However, membrane active peptides on the bacterial action and mechanism of cancer cells is inconclusive. Therefore, it has important theoretical and practical significance to study mechanism of the membrane active peptides. Membrane active peptide V13K subject to the preliminary work obtained as the parent peptide, using hydrophobic and different charge properties of amino acids between two groups of polypeptides with different hydrophobicity and charge properties of the design. First, by using twice dilution method on different detection polypeptide of gram positive bacteria and gram negative bacterial anti bacterial activity, according to the different cancer cells and the toxicity of anticancer activity to normal cells and red blood cells, the structure-activity relationship analysis of membrane active peptides with biological activity Peptide hydrophobicity and charge is closely related to the hydrophobic property and the ability of anti bacteria and stronger anticancer bioactive peptide; amino acid containing negative charge is relatively weak. At the same time, experiments confirmed that destruction membrane active peptides on bacteria and cancer cells than in normal cells and destruction of red blood cells. Then the permeability test of bacteria, the outer and inner membrane active peptides by flow cytometry, and the destructive effect on the cancer cell membrane. The results confirmed that the cell membrane active peptides on bacteria and cancer cells have ability to destroy the cell membrane. Moreover, the stronger the water hydrophobic polypeptide on bacteria and cancer cells the destruction of the stronger. In addition, membrane active peptides containing negative charged amino acid on bacteria and cancer cell destruction ability is relatively weak, this result is consistent with the result of biological activity and membrane active peptides. Further The simulation method by using phospholipid membrane, tryptophan blue shift and iodide quenching experiment experimental study of membrane active peptides and cell membrane phospholipid interactions. The results confirmed that the role of peptide and phospholipid analog membrane hydrophobic strong; peptide and phospholipid membranes containing negatively charged amino acid analog function is weak. In addition, we also found that the capacity of membrane active polypeptides containing positive charge and prokaryotic cells containing negative charge simulation film and cancer cell membrane simulation ability is relatively strong, while normal cell peptide and neutral phase of simulation of membrane is weak. This result is consistent with the result of biological activity and membrane active peptides and further proof of membrane active peptides with selective on bacterial cell membrane and cell membrane. Finally, the imaging technique of atomic force microscopy and single molecule force spectroscopy techniques, further research on membrane activity The mechanism of effect of different peptides on the cell membrane. The atomic force microscopy imaging technology can directly show the membrane active peptides with bacteria and cancer cells before and after the interaction of the cell membrane surface morphology. The size of the interaction of atomic force microscope singlemoleculeforcespectroscopy biofilm single molecule polypeptide with different phospholipid membrane and its corresponding simulation technology research through comparing and analyzing the effect of different membrane active peptides on the cell membrane of the magnitude and probability of effect, confirmed the ability of peptide cell membrane effect of the hydrophobic property of the stronger, and with negative charge amino acid polypeptide capability is weak. In addition, the capacity of membrane active peptides and prokaryotic cell membrane and cell membrane strong ability of normal cell membrane is relatively weak. The above results further verify the membrane active peptides targeting mechanism mainly depends on the membrane phospholipid composition difference ISO, which contains a positively charged polypeptide with negative charge of prokaryotic cells and cancer cells the ability of cell membrane is stronger, the results with the previous proposed "film mechanism of distinguishing is consistent. The membrane active peptides on different cell membrane targeting specificity, for further improving the design for bacteria and cancer cell membrane targeting peptide therapy and its clinical application lays an important theoretical basis.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R91

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