螺旋藻抗肿瘤肽的分离及壳聚糖纳米粒子复合物的制备

发布时间：2018-06-13 05:01

本文选题：螺旋藻 + 抗肿瘤肽　；参考：《华南理工大学》2012年硕士论文

【摘要】：螺旋藻含有大量蛋白质、丰富均衡的营养成分和多种生物活性物质，是一种优良的纯天然食品，被联合国粮农组织誉为“全球人类最理想的食品”。近年来，国内外人员研究表明，螺旋藻具有抗疲劳、抗辐射、抗病毒、抑制肿瘤、抗过敏、增强免疫力等多种功能，使其成为研究的热点。本文以蛋白质含量丰富的螺旋藻粉末为原料，采用超声破碎和反复冻融的方法提取螺旋藻蛋白。用不同蛋白酶对其进行水解，超滤获得不同分子量水解液。用MTT法筛选具有较好抗肿瘤活性的水解液，并用葡聚糖凝胶柱分离纯化，将纯化后的抗肿瘤肽继续进行抗肿瘤活性筛选。将最后得到活性高的抗肿瘤肽与壳聚糖相互作用，形成抗肿瘤肽-壳聚糖纳米粒子复合物，并对纳米粒子复合物进行了一系列表征。其结果如下：（1）以水解度为指标，用L_9（3~4）正交实验确定了不同蛋白酶优化水解条件：胰蛋白酶水解过程中最优条件是温度42℃，pH=8，酶与底物的比3%，水解度为38.49%；碱性蛋白酶水解过程中最优条件是pH=8.5，温度50℃，酶与底物的比5%，水解度为31.16%；胃蛋白酶水解过程中最优条件是温度37℃，pH=2，酶与底物的比为6%，水解度为7.07%；木瓜蛋白酶水解过程中最优条件是温度55℃，pH=6.5，酶与底物的比4%，水解度为27.80%。（2）用MTT法研究了不同酶水解螺旋藻蛋白的不同分子量水解液对人乳腺癌细胞（MCF-7）和肝癌细胞(HepG-2)体外生长抑制作用。结果显示：当药物终浓度均为1mg/mL时，胰蛋白酶水解产物5-10KD多肽对人乳腺癌细胞（MCF-7）和肝癌细胞(HepG-2)体外生长抑制率分别为45.19%，23.5%。（3）将胰蛋白酶5-10KD水解液经过葡聚糖凝胶柱（Sephadex G-50）分离纯化。最终确定洗脱条件为：洗脱速度为0.5mL/min，上样体积为2mL，洗脱液为超纯水，检测波长为215nm。按照出峰顺序共收集到三个峰。用福林酚法测得其肽浓度含量分别为57%，，41%和50%。（4）用MTT法对分离纯化的三个组分进行对肿瘤细胞体外生长抑制作用研究表明：当浓度均500μg/mL时，三种组分对人乳腺癌细胞（MCF-7）的体外生长抑制率分别为86%、98%和6%。对肝癌细胞（HepG-2）体外生长抑制率分别为76%、95%和28%。其中组分Y_2对两株细胞体外生长抑制作用最强，测得其对人乳腺癌细胞（MCF-7）和肝癌细胞（HepG-2）的IC50分别为61.36和60.96μg/mL。（5）采用三聚磷酸钠（TPP）离子交联法制备壳聚糖纳米粒子（CS NPs），确定了制备壳聚糖纳米粒子的合适TPP浓度（0.1-0.5mg/mL），并对组分Y_2包裹，制备壳聚糖纳米粒子复合物（Y_2-CS NPs）。结果显示：随着TPP浓度的增大体系的粒径逐渐增大。当浓度为0.5mg/mL时，体系最稳定，Zeta电位为41.5mv，平均粒径为152.7nm。并采用多种表征方法，验证了抗肿瘤肽壳聚糖纳米粒子复合物的成功制备。（6）研究了不同浓度Y_2对Y_2-CS NPs的包封率和载药量的影响。结果如下：Y_2-CSNPs的包封率随着组分Y_2浓度的增大而降低，当组分Y_2浓度由0.2mg/mL增加到1mg/mL时，包封率由42%下降到19%；而载药量出现了缓慢上升，由5%上升到15%。（7）采用MTT法，研究了壳聚糖纳米粒子（CS NPs）和抗肿瘤肽壳聚糖纳米粒子复合物（Y_2-CS NPs）对人乳腺癌细胞MCF-7和人肝癌细胞HepG-2体外生长抑制情况。结果表明：在CS NPs浓度为50-100μg/mL时候，对MCF-7细胞体外生长有一定的抑制作用，最高可达16.95%。Y_2-CS NPs体系对MCF-7细胞体外生长具有一定的抑制作用，抑制率最高为26.05%；CS NPs和Y_2-CS NPs体系对HepG-2细胞体外生长几乎没有抑制作用。
[Abstract]:Spirulina contains a large number of proteins, rich and balanced nutrients and a variety of bioactive substances. It is a good pure natural food. It is known as "the most ideal food in the world" by the United Nations Food and agricultural organization. In recent years, domestic and foreign researchers have shown that Spirulina has anti fatigue, radiation resistance, antiviral, anti-tumor, anti allergy, enhancement. Immunity and other functions make it a hot topic of research.
Spirulina powder with rich protein content was used as raw material to extract Spirulina protein by ultrasonic crushing and repeated freezing and thawing. Different protease was used to hydrolyze it and ultrafiltration to obtain different molecular weight hydrolysates. The hydrolysates with good antitumor activity were screened by MTT method and purified with glucan gel column. After the anti-tumor peptide continued to be screened for anti-tumor activity, the antitumor peptide and chitosan were interacted to form an antitumor peptide chitosan nanoparticle complex, and a series of characterization of the nanoparticles complex was carried out. The results are as follows:
(1) the optimum hydrolysis conditions of different proteases were determined by L_9 (3~4) orthogonal experiment. The optimum conditions for trypsin hydrolysis were temperature 42, pH=8, the ratio of enzyme to substrate by 3%, and the degree of hydrolysis of 38.49%, and the optimum conditions for the hydrolysis of alkaline protease were pH=8.5, the temperature 50, the ratio of enzyme to substrate, and the degree of hydrolysis 31.16%. The optimum conditions for the hydrolysis of pepsin were temperature 37, pH=2, the ratio of enzyme to substrate was 6%, and the degree of hydrolysis was 7.07%. The optimum conditions for the hydrolysis of papain were temperature 55, pH=6.5, the ratio of enzyme to substrate, and the degree of hydrolysis was 27.80%.
(2) the inhibitory effects of different molecular weight hydrolysates of different enzyme hydrolysates on human breast cancer cells (MCF-7) and hepatoma cells (HepG-2) in vitro were studied by MTT method. The results showed that when the final concentration of the drug was 1mg/mL, the 5-10KD polypeptide of trypsin hydrolysate to human breast cancer cell (MCF-7) and liver cancer cell (HepG-2) exogeny The rate of long inhibition was 45.19%, 23.5%.
(3) separation and purification of trypsin 5-10KD hydrolysate through dextran gel column (Sephadex G-50). The elution conditions were determined as follows: the elution rate was 0.5mL/min, the sample volume was 2mL, the eluant was super pure water, and the detection wavelength was 215nm. according to the peak order. The concentration of the peptide was 57%, 41% respectively. And 50%.
(4) the inhibitory effect of three components of the isolated and purified components on the growth of the tumor cells in vitro by MTT method showed that the inhibitory rate of three components to human breast cancer cells (MCF-7) in vitro was 86%, 98% and 6%. were 76%, 95% and 28%., respectively, when the concentration was 500 mu, respectively, and 95% and 28%.. The two cells had the strongest inhibitory effect on growth in vitro, and the IC50 of human breast cancer cells (MCF-7) and hepatoma cells (HepG-2) was 61.36 and 60.96 g/mL. respectively.
(5) chitosan nanoparticles (CS NPs) were prepared by sodium tripolyphosphate (TPP) ion crosslinking method. The appropriate TPP concentration (0.1-0.5mg/mL) of chitosan nanoparticles was determined. The chitosan nanoparticles complex (Y_2-CS NPs) was prepared by wrapping the component Y_2. The results showed that the particle size of the chitosan nanoparticles (Y_2-CS NPs) was increased with the increase of TPP concentration. At 0.5mg/mL, the system was the most stable, the Zeta potential was 41.5mv, the average particle size was 152.7nm. and a variety of characterization methods were used to verify the successful preparation of the antitumor peptide chitosan nanoparticles complex.
(6) the effects of different concentrations of Y_2 on the encapsulation efficiency and drug loading of Y_2-CS NPs are studied. The results are as follows: the encapsulation efficiency of Y_2-CSNPs decreases with the increase of the component Y_2 concentration. When the component Y_2 concentration increases from 0.2mg/mL to 1mg/mL, the encapsulation efficiency decreases from 42% to 19%, while the drug loading increases slowly from 5% to 15%..
(7) the inhibitory effect of chitosan nanoparticles (CS NPs) and antitumor peptide chitosan nanoparticles complex (Y_2-CS NPs) on the growth of human breast cancer cell MCF-7 and human hepatocarcinoma cell HepG-2 in vitro was studied by MTT method. The results showed that the maximum inhibitory effect on the growth of MCF-7 cells in vitro was certain when the NPs concentration of CS NPs was 50-100 micron g/mL. The 16.95%.Y_2-CS NPs system has a certain inhibitory effect on the growth of MCF-7 cells in vitro, the highest inhibition rate is 26.05%, and CS NPs and Y_2-CS NPs system have almost no inhibitory effect on the growth of HepG-2 cells in vitro.
【学位授予单位】：华南理工大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：TS201.2;R151

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