【作者】 王鹤；

【导师】 董维兵；

【作者基本信息】 辽宁师范大学 ， 细胞生物学， 2020， 硕士

【摘要】 基因疗法作为一种解决人类遗传疾病的潜在有效方法引起了人们的极大关注。基因载体分为病毒载体和非病毒载体,病毒载体转染效率高但制备起来比较困难较难获得、传递的基因容量较小、特异靶向性能低、有较强的免疫原性、容易产生变异病毒等缺点,因此极大的限制了其在临床上的应用。目前研制安全有效且价格低廉、易实现规模化生产的非病毒基因传递系统是基因治疗的关键。在本文中,我们选择抗菌肽L-K6作为模板进行改造,L-K6是以中国林蛙的皮肤分泌物中分离纯化的天然抗菌肽Temporin-1 CEb为模板得到的改造肽,含有Trp的抗菌肽与癌症细胞膜有良好的破坏作用,可以透过细胞膜与胞内物质发生相互作用。在前期工作基础上,通过在不同位置进行单个Trp残基的替换设计合成了一系列含有Trp的新型抗菌肽I1W、L5W、L12W,发现它们细胞亲和性增高、疏水性增加,推测其可能具有良好的转染能力,因此在本论文中,我们探究了抗菌肽L-K6及其类似物与外源DNA结合的能力,及其细胞内转染性能。首先,通过圆二色谱(CD)实验检测了多肽L-K6、I1W、L5W和L12W与DNA相互作用后的二级结构,结果发现与L-K6相比,I1W、L5W、L12W的α螺旋含量均有明显的提高,I1W、L5W、L12W和L-K6的α螺旋含量分别为:67.68%、44.14%、42.91%和36.99%;动态光散射检测多肽分子粒径与多肽Zeta电位测定结果表明L-K6、L12W、L5W、I1W的粒径逐个变小;电位升高;并通过CCK-8细胞毒实验检测了四种多肽对人宫颈癌Hela细胞和小鼠肾上皮293T细胞的抑制作用,结论说明当四种多肽浓度低于25μM时对人宫颈癌细胞和小鼠肾上皮细胞的生长影响较小;细胞活力高于90%,因此在转染实验中选用25μM浓度以下的多肽可以排除多肽对细胞生长的影响。为了研究多肽与DNA能否融合与融合方式,琼脂糖凝胶电泳实验结果表明L-K6及其类似物均能与ct DNA结合。动态光散射实验表明抗菌肽L-K6、I1W、L5W和L12W与ct DNA在质量比为1:1时的粒径依次为126.4nm、118.7nm、112.9nm和86.4nm均低于200nm这样大小的复合物有利于细胞对其的摄取。L-K6及其改造肽与ct DNA复合物的Zeta电位在质量比为0.25:1时能够对DNA表面负电荷发生中和反应,其Zeta电位呈正值,并且随着多肽与DNA质量比的增加电位随之增加。紫外光谱法和圆二色谱等实验表明L-K6系列肽与ct DNA结合方式均为静电结合,结合能力与多肽与DNA质量比呈正相关。我们对抗菌肽L-K6及其类似物作为非病毒基因载体的转染GFP-1质粒的效率进行了研究。首先我们通过溶血实验证明了L-K6及其类似物对细胞无毒性影响;激光扫描共聚焦显微镜观察到GFP-1表达的绿色荧光蛋白在人宫颈癌细胞和小鼠肾上皮细胞内和细胞核内均有明显分布;并采用流式细胞术检测GFP-1表达的绿色荧光蛋白在人宫颈癌细胞和小鼠肾上皮细胞表达效率;细胞中质粒基因GFP-1表达绿色荧光蛋白的水平呈现以下趋势L-K6<L12W<L5W<I1W。通过荧光酶标仪进一步强调了L-K6系列多肽与DNA质量比越高转染效率越高,与商用的Lipo2000相比I1W与L5W转染的荧光强度提高了百分之五左右,说明通过以抗菌肽L-K6为模板,在不同位置进行单个Trp残基的替换可以提高基因转染效率。更多还原

【Abstract】 Gene therapy as a potential effective method to solve human genetic diseases have arouse great concern.Gene vectors are divided into viral vectors and non-viral vectors.Viral vectors have some disadvantages,such as high transfection efficiency but difficult preparation,small target gene capacity,poor targeting specificity,strong immunogenicity,easy to produce wild type virus and so on.Therefore,its clinical application is greatly limited.At present,the key to gene therapy is to develop a safe,effective and cheap non viral gene delivery system.In this paper,we selected the antibacterial peptide L-K6 as a template for modification.L-K6 is a modified peptide obtained from the natural antibacterial peptide Temporin-1 CEb which is isolated and purified from the skin secretions of Rana chensinensis.The antimicrobial peptide containing Trp has a good killing effect on cancer cell membranes,and can interact with intracellular substances through cell membranes.Based on the previous work,a series of new antibacterial peptides I1 W,L5W,and L12 W containing Trp were synthesized by replacing and designing single Trp residues at different positions.It was found that their cell affinity and hydrophobicity were increased,and it was speculated that they may have good transfection ability.So in this paper,we explored the ability of antibacterial peptide L-K6 and its analogs to bind to exogenous GFP-1 DNA,and its intracellular transfection performance.First,the secondary structure of the peptides L-K6,I1 W,L5W,and L12 W interacting with DNA is detected by circular dichroism(CD)experiments.The results show that compared with L-K6,the α-helix content of I1 W,L5W,L12 W.The α-helix contents of I1 W,L5W,L12 W and L-K6 are: 67.68%,44.14%,42.91% and 36.99% respectively;the results of dynamic light scattering detection of peptide molecular size and peptide Zeta potential show that the particle size of L-K6,L12 W,L5W,and I1 W become smaller one by one;the potential increased;and the inhibitory effect of four peptides on human cervical cancer cells and mouse renal epithelial cells was tested by CCK8 experiment.When it is lower than 25μM,it has little effect on the growth of human cervical cancer cells and mouse kidney epithelial cells;the cell viability is higher than 90%,so selecting peptides with a concentration of less than 25μM in the transfection experiment can exclude the effect of peptides on cell growth.In order to study the binding mode and ability of peptides and DNA,the results of agarose gel electrophoresis experiments show that L-K6,L12 W and ct DNA are completely bound when the mass ratio is 1: 1,while I1 W and L5 W were completely bound to ct DNAwhen the mass ratio is 0.5: 1,indicating that L-K6 and its analogs can bind to ct DNA.are126.4nm,118.7nm,112.9nm and 86.4nm all of which are below 200 nm,the substance is beneficial to the uptake of cells.The Zeta potential of L-K6 and its analogs with ct DNA complex can neutralize the negative charge on the surface of DNA when the mass ratio is 0.25:1,and its zeta potential becomes positive,and the zeta potential increases with the increase of the mass ratio of peptide to DNA.The experiments of UV spectrum and circular dichroism indicate that the combination of L-K6 series peptides and ct DNA is electrostatically combined,and the binding capacity is positively related to the mass ratio of peptide to DNA.We study the efficiency of transfection of GFP-1 plasmid with antibacterial peptide L-K6 and its analogs as non-viral gene vectors.First,we prove that L-K6 and its analogs have no toxic effect on cells through hemolysis experiments;The GFP-1 expressed in human cervical cancer cells and mouse renal epithelial cells is observed by laser scanning confocal microscopy;flow cytometry is used to detect the expression efficiency of GFP-1 expressed green fluorescent protein in human cervical cancer cells and mouse kidney epithelial cells;the level of plasmid gene GFP-1 expressed green fluorescent protein in the cells show the following trends: L-K6 <L12W <L5W <I1W.The fluorescence microplate reader further show that the higher the mass ratio of L-K6 series peptides to DNA,the more obvious transfection effect.Compared with the commercial Lipo2000,the fluorescence intensity of I1 W and L5 W transfection increas by about 5%,indicating that the efficiency of gene transfection can be improved by using antimicrobial peptide L-K6 as template and replacing single Trp residues at different locations.更多还原