本文選題：螺旋藻 + 抗腫瘤肽　； 參考：《華南理工大學(xué)》2012年碩士論文

【摘要】：螺旋藻含有大量蛋白質(zhì)、豐富均衡的營養(yǎng)成分和多種生物活性物質(zhì)，是一種優(yōu)良的純天然食品，被聯(lián)合國糧農(nóng)組織譽(yù)為“全球人類最理想的食品”。近年來，國內(nèi)外人員研究表明，螺旋藻具有抗疲勞、抗輻射、抗病毒、抑制腫瘤、抗過敏、增強(qiáng)免疫力等多種功能，使其成為研究的熱點(diǎn)。 本文以蛋白質(zhì)含量豐富的螺旋藻粉末為原料，采用超聲破碎和反復(fù)凍融的方法提取螺旋藻蛋白。用不同蛋白酶對其進(jìn)行水解，超濾獲得不同分子量水解液。用MTT法篩選具有較好抗腫瘤活性的水解液，并用葡聚糖凝膠柱分離純化，將純化后的抗腫瘤肽繼續(xù)進(jìn)行抗腫瘤活性篩選。將最后得到活性高的抗腫瘤肽與殼聚糖相互作用，形成抗腫瘤肽-殼聚糖納米粒子復(fù)合物，并對納米粒子復(fù)合物進(jìn)行了一系列表征。其結(jié)果如下： （1）以水解度為指標(biāo)，用L_9（3~4）正交實(shí)驗(yàn)確定了不同蛋白酶優(yōu)化水解條件：胰蛋白酶水解過程中最優(yōu)條件是溫度42℃，pH=8，酶與底物的比3%，水解度為38.49%；堿性蛋白酶水解過程中最優(yōu)條件是pH=8.5，溫度50℃，酶與底物的比5%，水解度為31.16%；胃蛋白酶水解過程中最優(yōu)條件是溫度37℃，pH=2，酶與底物的比為6%，水解度為7.07%；木瓜蛋白酶水解過程中最優(yōu)條件是溫度55℃，pH=6.5，酶與底物的比4%，水解度為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對人乳腺癌細(xì)胞（MCF-7）和肝癌細(xì)胞(HepG-2)體外生長抑制作用。結(jié)果顯示：當(dāng)藥物終濃度均為1mg/mL時(shí)，胰蛋白酶水解產(chǎn)物5-10KD多肽對人乳腺癌細(xì)胞（MCF-7）和肝癌細(xì)胞(HepG-2)體外生長抑制率分別為45.19%，23.5%。 （3）將胰蛋白酶5-10KD水解液經(jīng)過葡聚糖凝膠柱（Sephadex G-50）分離純化。最終確定洗脫條件為：洗脫速度為0.5mL/min，上樣體積為2mL，洗脫液為超純水，檢測波長為215nm。按照出峰順序共收集到三個(gè)峰。用福林酚法測得其肽濃度含量分別為57%，，41%和50%。 （4）用MTT法對分離純化的三個(gè)組分進(jìn)行對腫瘤細(xì)胞體外生長抑制作用研究表明：當(dāng)濃度均500μg/mL時(shí)，三種組分對人乳腺癌細(xì)胞（MCF-7）的體外生長抑制率分別為86%、98%和6%。對肝癌細(xì)胞（HepG-2）體外生長抑制率分別為76%、95%和28%。其中組分Y_2對兩株細(xì)胞體外生長抑制作用最強(qiáng)，測得其對人乳腺癌細(xì)胞（MCF-7）和肝癌細(xì)胞（HepG-2）的IC50分別為61.36和60.96μg/mL。 （5）采用三聚磷酸鈉（TPP）離子交聯(lián)法制備殼聚糖納米粒子（CS NPs），確定了制備殼聚糖納米粒子的合適TPP濃度（0.1-0.5mg/mL），并對組分Y_2包裹，制備殼聚糖納米粒子復(fù)合物（Y_2-CS NPs）。結(jié)果顯示：隨著TPP濃度的增大體系的粒徑逐漸增大。當(dāng)濃度為0.5mg/mL時(shí)，體系最穩(wěn)定，Zeta電位為41.5mv，平均粒徑為152.7nm。并采用多種表征方法，驗(yàn)證了抗腫瘤肽殼聚糖納米粒子復(fù)合物的成功制備。 （6）研究了不同濃度Y_2對Y_2-CS NPs的包封率和載藥量的影響。結(jié)果如下：Y_2-CSNPs的包封率隨著組分Y_2濃度的增大而降低，當(dāng)組分Y_2濃度由0.2mg/mL增加到1mg/mL時(shí)，包封率由42%下降到19%；而載藥量出現(xiàn)了緩慢上升，由5%上升到15%。 （7）采用MTT法，研究了殼聚糖納米粒子（CS NPs）和抗腫瘤肽殼聚糖納米粒子復(fù)合物（Y_2-CS NPs）對人乳腺癌細(xì)胞MCF-7和人肝癌細(xì)胞HepG-2體外生長抑制情況。結(jié)果表明：在CS NPs濃度為50-100μg/mL時(shí)候，對MCF-7細(xì)胞體外生長有一定的抑制作用，最高可達(dá)16.95%。Y_2-CS NPs體系對MCF-7細(xì)胞體外生長具有一定的抑制作用，抑制率最高為26.05%；CS NPs和Y_2-CS NPs體系對HepG-2細(xì)胞體外生長幾乎沒有抑制作用。
[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.
【學(xué)位授予單位】：華南理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：TS201.2;R151

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