微藻硫酸酯化复合多糖的抗肿瘤及免疫调节活性研究

发布时间：2018-07-21 09:34

【摘要】：微藻的生长条件和环境特点决定了其胞内多糖具有一些有别于其它生物多糖的结构和功能,不同来源的微藻多糖具有不同的生物活性,并且多糖分子中的硫酸基团的含量对其生物活性有明显的影响。如果对多种微藻多糖进行硫酸酯化制备,并进行相应的复合可筛选出最佳比例复合多糖,这对今后微藻复合多糖的开发及广泛应用有着积极地借鉴意义和促进作用。本论文以螺旋藻纯多糖(PSP)和小球藻纯多糖(CSP)为研究对象,采用浓硫酸法将两种多糖进行了硫酸酯化的制备,并将两种硫酸酯化多糖按照一定比例配置成15种不同比例的硫酸酯化微藻复合多糖,通过MTT法分别对这15种不同的硫酸酯化复合多糖进行了体外抗肿瘤的筛选,并采用体外细胞毒性试验测定了最佳比例多糖的最大无毒浓度。结果表明,硫酸酯化后的螺旋藻多糖(SPSP)和硫酸酯化后的小球藻多糖(SCSP)的SO42-含量和取代度值分别为19.58%、0.42和19.30%、0.41,傅里叶红外光谱扫描图显示,两种硫酸酯化多糖不但保存了原多糖的结构而且硫酸基团被成功的添加到多糖分子中。15种不同比例的微藻硫酸酯化复合多糖中SCP12的体外抗肿瘤效果最好,抑制率达到52.31 %,最大无毒浓度为6.4 mg/mL。采用MTT法测定硫酸酯化复合多糖SCP12的体外抗肿瘤活性,并以市场购买的药用PSP作为对照,结果表明,SCP12对肿瘤细胞的生长抑制作用明显,且抑制作用明显高于市售PSP,在设定的浓度范围内(0.4-6.4mg/mL)呈现出浓度依赖性,当浓度达到6.4 mg/mL时,SCP12对乳腺癌细胞(MCF-7)、肝癌细胞(HepG2)和肺癌细胞(A549)的抑制率分别为52.31%、39.71%和16.15%;将乳腺癌细胞转接到小鼠体内,给予不同剂量治疗,结果显示,高、中、低(0.8、3.2、6.4mg/mL)三个剂量组的SCP12均可抑制乳腺癌细胞的生长,其抑制率分别为82.19%、71.32%和28.77%; SCP12体外抗凝血结果显示,高、中、低(0.8、3.2、6.4mg/mL)的三个剂量组的SCP12均可改善TT、PT和APTT的值,说明SCP12具有抗凝血作用。硫酸酯化复合多糖SCP12体外免疫调节活性研究表明,在0.4-6.4 mg/mL浓度范围内,SCP12能显著提高小鼠腹腔巨噬细胞释放NO的能力、促进小鼠脾淋巴细胞的增殖,其效果明显高于对照组单糖PSP,但对小鼠腹腔巨噬细胞吞噬中性红的能力促进作用一般;SCP12体内对二硝基氟苯(DNFB)诱导的小鼠迟发变态反应的结果显示,高、中、低剂量组耳肿胀度明显增加,说明SCP12具有增强因DNFB引起的小鼠迟发型变态反应。以上结果表明,硫酸酯化复合多糖SCP12具有明显的免疫调节作用。
[Abstract]:The growth conditions and environmental characteristics of microalgae determined that the intracellular polysaccharides had some different structures and functions from other biological polysaccharides, and the microalgae polysaccharides from different sources had different biological activities. The content of sulphate groups in polysaccharides has a significant effect on its biological activity. If a variety of microalgae polysaccharides were prepared by sulfate esterification and the corresponding compound could be selected the best proportion of the composite polysaccharides could be selected which would be helpful to the development and wide application of microalgae polysaccharides in the future. In this paper, the pure polysaccharide of Spirulina platensis (PSP) and the pure polysaccharide of Chlorella vulgaris (CSP) were prepared by concentrated sulfuric acid method. Two kinds of sulfated polysaccharides were configured into 15 different proportions of sulfated microalgae polysaccharides according to a certain proportion. The 15 kinds of sulfated polysaccharides were screened by MTT method in vitro. The maximum nontoxic concentration of the optimum proportion of polysaccharides was determined by in vitro cytotoxicity test. The results showed that the SO42- content and degree of substitution of sulfated polysaccharide (SPSP) and chlorella polysaccharide (SCSP) were 19.58 and 19.300.41, respectively. The two sulfated polysaccharides not only preserved the structure of the original polysaccharides, but also the sulfate groups were successfully added to the polysaccharide molecules. 15 different proportions of microalgae sulfated polysaccharides with SCP12 had the best anti-tumor effect in vitro. The inhibition rate was 52.31 and the maximum nontoxic concentration was 6.4 mg / mL. MTT assay was used to determine the antitumor activity of sulfated compound polysaccharide SCP12 in vitro. The results showed that SCP12 had obvious inhibitory effect on the growth of tumor cells. The inhibitory effect was significantly higher than that of PSPs in a concentration dependent manner (0.4-6.4 mg / mL). When the concentration reached 6.4 mg / mL, the inhibition rates of SCP12 on MCF-7, HepG2 and A549 cells were 52.31% and 16.15%, respectively. The growth of breast cancer cells was inhibited by SCP12 in three dose groups (0.8-3.2-6.4mg / mL), the inhibitory rates were 82.191.32% and 28.77mg / mL, respectively. The results of in vitro anticoagulant of SCP12 showed that SCP12 of high, middle and low dose groups (0.83.26.4mg / mL) could improve the values of TTPT and APTT. It indicated that SCP 12 had anticoagulant effect. The immunoregulatory activity of sulfated polysaccharide SCP12 was studied in vitro. The results showed that SCP12 could significantly improve the ability of releasing no from peritoneal macrophages and promote the proliferation of splenic lymphocytes in mice in the concentration range of 0.4-6.4 mg / mL. Its effect was significantly higher than that of control group, but the effect of SCP12 on phagocytosis of neutral red in peritoneal macrophages of mice was higher and higher than that of DNFB induced delayed allergic reaction in mice. The ear swelling in low dose group was significantly increased, indicating that SCP 12 enhanced delayed allergic reaction induced by DNFB in mice. The results showed that the sulfated compound polysaccharide SCP 12 had obvious immunomodulatory effect.
【学位授予单位】：天津科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96

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