α-螺旋型膜活性多肽对模拟膜和生物膜作用机理的研究

发布时间：2018-01-16 04:06

本文关键词：α-螺旋型膜活性多肽对模拟膜和生物膜作用机理的研究　出处：《吉林大学》2017年博士论文　论文类型：学位论文

【摘要】：在临床治疗中,细菌感染一直是备受人们关注的问题。抗生素的出现有效抑制各种致病细菌、真菌的感染,对保护人类的健康起着重要的作用。但是,抗生素的滥用导致耐药菌甚至是超级细菌的产生,为人类带来新的问题。因此,寻找抗生素的合适替代品成为人们研究的热点。同时,癌症作为一种常见的恶性疾病,发病率呈现逐年升高的趋势,严重威胁着人类的健康和生命。目前临床使用的抗肿瘤药物主要是烷基化试剂和抗代谢类药物,很多药物不仅利用率低,而且具有很强的毒副作用。因此,寻找新型的抗肿瘤药物也具有十分重要的意义。膜活性多肽是一类作用于细胞膜的生物活性肽,主要包括抗细菌肽、抗真菌肽和抗癌肽。膜活性多肽以其广谱性、速效性和不易产生耐药性等特点而受到人们的广泛重视。但是,膜活性多肽对细菌和癌细胞作用机理尚无定论。因此,研究膜活性多肽的作用机理具有重要的理论和实际意义。本课题以前期工作获得的膜活性多肽V13K为母肽,利用氨基酸之间疏水性和电荷不同的性质,改造设计两组具有不同疏水性和电荷性质的多肽。首先,通过采用倍比稀释法检测多肽对不同的革兰氏阳性细菌和阴性细菌的抗细菌活性、针对不同的癌细胞的抗癌活性以及针对正常细胞和血红细胞的毒性,通过构效关系分析得到膜活性多肽的生物活性与多肽的疏水性和电荷密切相关,疏水性越强,其抗细菌和抗癌的能力越强;含有负电荷氨基酸的多肽生物活性相对较弱。同时,实验证实膜活性多肽对细菌和癌细胞的破坏能力要高于对正常细胞和血红细胞的破坏能力。其次,通过膜活性多肽对细菌外膜和内膜渗透性试验,和流式细胞仪检测其对癌细胞膜的破坏作用。结果证实,膜活性多肽对细菌和癌细胞的细胞膜均具有破坏能力。并且,疏水性越强的多肽对细菌和癌细胞的细胞膜破坏能力越强。此外,含有负电荷氨基酸的膜活性多肽对细菌和癌细胞的细胞膜破坏能力相对较弱,这一结果与膜活性多肽的生物活性实验结果相一致。进一步利用磷脂模拟膜方法,通过色氨酸蓝移实验和碘化钾淬灭实验研究膜活性多肽与细胞膜磷脂成分相互作用的过程。结果证实,疏水性强的多肽与磷脂模拟膜的作用能力强;含有负电荷氨基酸的多肽与磷脂模拟膜的作用能力较弱。此外,我们还发现含有正电荷的膜活性多肽与含有负电荷的原核细胞模拟膜和癌细胞模拟膜的作用能力相对较强,而多肽与中性的正常细胞模拟膜的作用能力相对较弱。这一结果与膜活性多肽的生物活性实验结果相一致,进一步证明膜活性多肽对细菌细胞膜和癌细胞膜具有选择性。最后,通过原子力显微镜成像技术和单分子力谱技术,进一步研究膜活性多肽对不同细胞膜作用的机理。其中,原子力显微镜成像技术可以直观的展现膜活性多肽与细菌和癌细胞相互作用前后的细胞膜表面形貌。原子力显微镜单分子力谱技术研究单分子多肽与不同磷脂模拟膜及其对应的生物膜的相互作用力大小。通过对比分析膜活性多肽对不同细胞膜作用的力值大小和作用几率,证实疏水性越强的多肽与细胞膜作用的能力越强,而含有负电荷氨基酸的多肽作用能力较弱。此外,利用膜活性多肽与原核细胞膜和癌细胞膜的作用能力较强,对正常细胞膜作用能力相对较弱。上述结果进一步验证膜活性多肽的靶向性作用机制主要依赖于细胞膜磷脂成分的差异,即含有正电荷的多肽与含有负电荷的原核细胞和癌细胞的细胞膜作用能力更强,这一结果与前期提出的“膜区分机理”相一致。膜活性多肽对不同细胞膜作用的靶向特异性,为进一步改造设计针对细菌和癌细胞靶向治疗的膜活性多肽及其临床应用奠定重要的理论基础。
[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.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R91

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