钝顶螺旋藻两个生态种多糖的抗菌、抗肿瘤活性及其机理的研究
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摘要
本论文以鄂尔多斯高原碱湖钝顶螺旋藻和非洲Chad湖钝顶螺旋藻为材料,分离、提取、纯化和鉴定了其多糖,并对多糖的抗菌、抗肿瘤活性及其作用机理进行了比较研究,以期为鄂尔多斯高原碱湖钝顶螺旋藻及其多糖产品的开发、新的抗肿瘤药物的筛选和寻找天然的抗生素替代品及动物免疫增强剂奠定理论和应用研究基础。论文研究内容分四部分:
(1)采用传统的水提醇沉法、三氯乙酸除蛋白法和活性炭脱色法提取并初步纯化了钝顶螺旋藻两个生态种多糖,SephadexG-100凝胶柱层析法进一步分离纯化得到了鄂尔多斯高原碱湖钝顶螺旋藻多糖ESP和非洲Chad湖钝顶螺旋藻多糖FSP。纸层析法、紫外光谱法、比旋光度法和SephadexG-100凝胶柱层析法检验ESP和FSP都为均一的多糖组分;低温冷冻干燥后ESP为乳白色絮状结晶,FSP为黄色絮状结晶,二者均为易溶于水的水溶性多糖。硫酸-苯酚法测定ESP和FSP的多糖含量分别为89.75%和62.87%;非糖成分检测发现ESP和FSP中残留的非糖成分主要为灰分,其次为少量蛋白质,初步推断这两种多糖均为蛋白结合多糖。
在上述研究基础上采用SephadexG-150凝胶柱层析、TLC、HPLC、IR、NMR等技术方法分别对ESP和FSP进行了分子量和结构的研究。结果:ESP和FSP的分子量分别为77625 D和85114 D。ESP糖链的连接方式主要为[α-Glc(1→4)-]n,还有少量的α-Glc(1→6)-、α-Fuc(1→2)-和β-Xyl(1→3)-,-α-Rha为末端糖基;构成其糖链的单糖主要是α-吡喃型的已糖,占比例最大的是葡萄糖,其次为鼠李糖和少量的阿拉伯糖、木糖、半乳糖、甘露糖和果糖。FSP糖链的连接方式为α-Glc(1→6)-和α-Xyl(1→2),-β-Xyl、-β-Gal和-α-Rha为末端糖基;构成其糖链的主要是α-或β-型的吡喃葡萄糖,其次是鼠李糖和少量的木糖、甘露糖、半乳糖和阿拉伯糖。
(2)采用管碟法研究了ESP和FSP这两种多糖的体外抗菌活性。结果:ESP和FSP均有抑菌作用,但抑制效果与多糖浓度、细菌种类有关。ESP在20 mg/mL和40 mg/mL添加量时均表现出对痢疾杆菌有较明显的抑制作用,抑菌圈分别为14.17 mm和12.16 mm。FSP表现为在20 mg/mL添加量时对痢疾杆菌、假单孢绿脓杆菌和变形杆菌有抑制作用,抑菌圈分别为12.13 mm、9.10 mm和9.07 mm。
(3)采用S180腹水瘤动物模型,考察了ESP和FSP对小鼠体内S180腹水瘤的抑制作用,同时采用MTT和ELISA等实验技术方法检测了ESP和FSP对肿瘤鼠脾淋巴细胞增殖及免疫细胞因子水平的影响,初步探讨了ESP和FSP对荷瘤小鼠免疫增强作用的分子机制。结果:ESP和FSP均能改善荷瘤小鼠的生存质量,抑制小鼠S180移植性肿瘤的生长,缩小肿瘤在体内的浸润范围,延长小鼠的生存时间,中剂量的作用效果最明显,对荷瘤小鼠的生命延长率分别达到50.67%和52.00%。对免疫功能的影响表现为,能在一定程度上降低肿瘤小鼠的脏器损伤,提高肿瘤小鼠的胸腺和脾脏指数,进而提高小鼠的免疫功能;在抑制肿瘤生长的同时,ESP和FSP还能够提高荷瘤小鼠血清IL-2、TNF-α和IFN-γ水平。在体外,ESP和FSP均能够促进ConA诱导的荷瘤小鼠脾淋巴细胞的增殖反应。ESP和FSP中剂量组的对荷瘤小鼠免疫细胞因子、脾淋巴细胞增殖和免疫器官指数的影响最明显。以上分析表明,适宜剂量的ESP和FSP对S180腹水型肿瘤有明显的抑制作用,并能增强荷瘤小鼠由T细胞主导的细胞免疫功能,提高机体的免疫调节作用,免疫调节可能是其抗肿瘤作用的机制之一。
(4)采用MTT法研究了ESP和FSP体外对小鼠肉瘤S180,小鼠白血病细胞L1210、人慢性髓性白血病细胞K562和小鼠淋巴瘤细胞YAC-1生长的影响;采用流式细胞术(flow cytometry,FCM)研究ESP和FSP对S180细胞周期和细胞凋亡的影响。结果:
ESP和FSP高、中、低三个剂量组对小鼠肉瘤细胞S180、小鼠白血病细胞L1210、人慢性髓性白血病细胞K562、小鼠淋巴瘤细胞YAC-1的体外增殖均有不同程度的抑制作用;但是,仅ESP和FSP中剂量组对K562的抑制率超过了30%,分别为35.88%和33.98%。ESP和FSP均表现出中剂量的对这4种肿瘤细胞体外增殖活性的抑制作用最明显,但其对S180、YAC-1和K562的抑制作用均显著低于CTX组(P<0.05)。与黄芪糖组相比,仅ESP和FSP中剂量组对K562和FSP中剂量组对YAC-1细胞增殖活性的抑制作用显著高于黄芪糖组(P<0.05)。
FCM结果显示,ESP和FSP均能引起细胞周期时相的改变,阻滞S180细胞于G1期,诱导细胞凋亡,并以中剂量组的作用效果最为明显(P<0.05)。
Extraction, isolation, purification and structural identification of polysaccharide from Spirulina (Arthrospira) platensis of alkaline lakes in Erdos Plateau and Chad Lake in Africa were studied, and also comparatively studied the mechanism of antitumor, activity of antibacteria and antitumor of the polysaccharide. The aim of this paper is to establish the basis of theoretical and applied research on developing polysaccharide of S. (A.) platensis of alkaline lakes in Erdos Plateau and its products, screening new antitumor drugs, natural alternatives to antibiotics and immunostimulant. The thesis is divided into four parts:
(1)Polysaccharide from S. (A.) platensis of two ecogeographic species were extracted with the traditional water extraction and alcohol precipitation method, and purified preliminarily with the trichloroacetic acid and active carbon method. The two sorts of Polysaccharide were further purified with SephadexG-100 gel column chromatography, ESP of polysaccharide from S. (A.) platensis of alkaline lakes in Erdos Plateau and FSP of polysaccharide from S. (A.) platensis of Chad Lake in Africa were achieved.
ESP and FSP were homogeneous component, which have been proved with Paper chromatography, UV spectrum, specific rotation and SephadexG-100 gel column chromatography methods. ESP was milky white flocculent crystal, FSP was light yellow flocculent crystal after freeze-drying by low temperature, and they were both soluble in water. The Polysaccharide′content of ESP and FSP were 89.75% and 62.87%, which have been determined with Sulfuric acid-phenol method. Non-polysaccharide components were mainly ash and protein in FSP and ESP, which have been verified too. ESP and FSP were polysaccharide combined protein possibly that were conjectured.
The molecular weight and structure of ESP and FSP were studied with SephadexG-150 gel column chromatography, TLC, HPLC, IR and NMR methods on the basis of above studies. The results showed that the molecular weight of ESP and FSP were 77625 Dr and 85114 Dr, the connection pattern of single sugar in ESP were mainly [α-Glc(1→4)-]n, including fewα-Glc(1→6)-,α-Fuc(1→2)-,β-Xyl(1→3), -α-Rha as ended glycosylation, The monosaccharide of constituting ESP′were mainlyα-pyranose, largest in glucose, followed by rhamnose and few in arabinose, xylose, galactose, mannose and fructose. The connection pattern of single sugar in FSP wereα-Glc(1→6)- andα-Xyl(1→2), -β-Xyl, -β-Gal and -α-Rha were ended glycosylation, the monosaccharide of constituting FSP were mainlyα- orβ- type glucopyranose, followed by rhamnose and few xylose, mannose, galactose and arabinose.
(2)The antibacterial function of ESP and FSP were studied in vitro with cup-plate method. The results showed that ESP and FSP had bactericidal activity, but inhibitory effect to bacteria was related to concentration of polysaccharide and species of bacteria. Inhibition of ESP was better significant to Shigella dysenteriae in the concentration of 20 mg/mL and 40 mg/mL, and the diameter of bacteriostasis circle were 14.17 millimeters and 12.16 millimeters. FSP had bactericidal activity to Shigella dysenteriae, Pseudomonas aeruginosa and Proteus vulgaris in the concentration of 20 mg/mL, the diameters of the inhibition zone were 12.13 millimeters, 9.10 millimeters and 9.07 millimeters.
(3)The inhibitory effect of ESP and FSP to S180 ascites tumor in mice has been studied with the animal model of S180 ascites tumor. And then the effect of ESP and FSP on proliferation of spleen lymphocyte cell and the concentration of immune cell factors in serum were detected with MTT and ELISA methods, the molecular mechanism of immune enhancement of ESP and FSP were studied preliminarily to tumor-bearing mice. The results showed that ESP and FSP could improve the survival quality of tumor-bearing mice, inhibit the growth of S180 transplanted in mice, reduce the scope of tumor invasion in the body, lengthening survival time of tumor-bearing mice. Effect of the medium dose of ESP and FSP was most significantly on the survival time of tumor-bearing mice, the lengthening rate of life were 50.67% and 52.00%. ESP and FSP could reduce injury of organ in tumor-bearing mice, raising the thymus and spleen index, thus increasing immune function, enhance the levels of IL-2, TNF-αand IFN-γin serum of tumor-bearing mice when inhibit growth of tumor. ESP and FSP could promote the proliferation of spleen lymphocyte cell induced by ConA In vitro. The influence of medium dose of ESP and FSP to cell factors, the proliferation of spleen lymphocyte and immune organ index were most significantly. The above results show that ESP and FSP of appropriate dose can inhibit S180 ascites tumor significantly, enhance T cell-driven immune function, improve the immune regulate function of body. Immune regulate effect may be one of the mechanisms of antitumor.
(4)The effect of ESP and FSP on growth of sarcoma 180, mouse leukemia cells L1210, human chronic myelogenous leukemia K562 and mouse lymphoma cells YAC-1 had been studied with MTT methods, meanwhile effect of ESP and FSP on cell cycle and cell apoptosis of S180 have been studied too with flow cytometry. The results showed that ESP and FSP of different dose all had inhabitation to different degrees on above tumor cells in vitro. But, only the medium doses of ESP and FSP had a inhibition rate more than 30%, reached 35.88% and 33.98%. The medium dose of ESP and FSP all had the more obviously inhibition to the cell proliferation on above tumor cells, but the inhibition effect were significantly lower than CTX group to the S180, YAC-1 and K562 (P<0.05). Comparison with Astragalus sugar group, only the medium dose of ESP and FSP to K562, the medium dose of FSP to YAC-1 had more significant inhabitation than Astragalus sugar group (P<0.05).
The results of FCM showed that, ESP and FSP could cause cell cycle changes, block S180 cells in the G1 phase, induce apoptosis, and the effect of the medium dose of ESP and FSP were most significant (P<0.05) comparison with each other.
(1)采用传统的水提醇沉法、三氯乙酸除蛋白法和活性炭脱色法提取并初步纯化了钝顶螺旋藻两个生态种多糖,SephadexG-100凝胶柱层析法进一步分离纯化得到了鄂尔多斯高原碱湖钝顶螺旋藻多糖ESP和非洲Chad湖钝顶螺旋藻多糖FSP。纸层析法、紫外光谱法、比旋光度法和SephadexG-100凝胶柱层析法检验ESP和FSP都为均一的多糖组分;低温冷冻干燥后ESP为乳白色絮状结晶,FSP为黄色絮状结晶,二者均为易溶于水的水溶性多糖。硫酸-苯酚法测定ESP和FSP的多糖含量分别为89.75%和62.87%;非糖成分检测发现ESP和FSP中残留的非糖成分主要为灰分,其次为少量蛋白质,初步推断这两种多糖均为蛋白结合多糖。
在上述研究基础上采用SephadexG-150凝胶柱层析、TLC、HPLC、IR、NMR等技术方法分别对ESP和FSP进行了分子量和结构的研究。结果:ESP和FSP的分子量分别为77625 D和85114 D。ESP糖链的连接方式主要为[α-Glc(1→4)-]n,还有少量的α-Glc(1→6)-、α-Fuc(1→2)-和β-Xyl(1→3)-,-α-Rha为末端糖基;构成其糖链的单糖主要是α-吡喃型的已糖,占比例最大的是葡萄糖,其次为鼠李糖和少量的阿拉伯糖、木糖、半乳糖、甘露糖和果糖。FSP糖链的连接方式为α-Glc(1→6)-和α-Xyl(1→2),-β-Xyl、-β-Gal和-α-Rha为末端糖基;构成其糖链的主要是α-或β-型的吡喃葡萄糖,其次是鼠李糖和少量的木糖、甘露糖、半乳糖和阿拉伯糖。
(2)采用管碟法研究了ESP和FSP这两种多糖的体外抗菌活性。结果:ESP和FSP均有抑菌作用,但抑制效果与多糖浓度、细菌种类有关。ESP在20 mg/mL和40 mg/mL添加量时均表现出对痢疾杆菌有较明显的抑制作用,抑菌圈分别为14.17 mm和12.16 mm。FSP表现为在20 mg/mL添加量时对痢疾杆菌、假单孢绿脓杆菌和变形杆菌有抑制作用,抑菌圈分别为12.13 mm、9.10 mm和9.07 mm。
(3)采用S180腹水瘤动物模型,考察了ESP和FSP对小鼠体内S180腹水瘤的抑制作用,同时采用MTT和ELISA等实验技术方法检测了ESP和FSP对肿瘤鼠脾淋巴细胞增殖及免疫细胞因子水平的影响,初步探讨了ESP和FSP对荷瘤小鼠免疫增强作用的分子机制。结果:ESP和FSP均能改善荷瘤小鼠的生存质量,抑制小鼠S180移植性肿瘤的生长,缩小肿瘤在体内的浸润范围,延长小鼠的生存时间,中剂量的作用效果最明显,对荷瘤小鼠的生命延长率分别达到50.67%和52.00%。对免疫功能的影响表现为,能在一定程度上降低肿瘤小鼠的脏器损伤,提高肿瘤小鼠的胸腺和脾脏指数,进而提高小鼠的免疫功能;在抑制肿瘤生长的同时,ESP和FSP还能够提高荷瘤小鼠血清IL-2、TNF-α和IFN-γ水平。在体外,ESP和FSP均能够促进ConA诱导的荷瘤小鼠脾淋巴细胞的增殖反应。ESP和FSP中剂量组的对荷瘤小鼠免疫细胞因子、脾淋巴细胞增殖和免疫器官指数的影响最明显。以上分析表明,适宜剂量的ESP和FSP对S180腹水型肿瘤有明显的抑制作用,并能增强荷瘤小鼠由T细胞主导的细胞免疫功能,提高机体的免疫调节作用,免疫调节可能是其抗肿瘤作用的机制之一。
(4)采用MTT法研究了ESP和FSP体外对小鼠肉瘤S180,小鼠白血病细胞L1210、人慢性髓性白血病细胞K562和小鼠淋巴瘤细胞YAC-1生长的影响;采用流式细胞术(flow cytometry,FCM)研究ESP和FSP对S180细胞周期和细胞凋亡的影响。结果:
ESP和FSP高、中、低三个剂量组对小鼠肉瘤细胞S180、小鼠白血病细胞L1210、人慢性髓性白血病细胞K562、小鼠淋巴瘤细胞YAC-1的体外增殖均有不同程度的抑制作用;但是,仅ESP和FSP中剂量组对K562的抑制率超过了30%,分别为35.88%和33.98%。ESP和FSP均表现出中剂量的对这4种肿瘤细胞体外增殖活性的抑制作用最明显,但其对S180、YAC-1和K562的抑制作用均显著低于CTX组(P<0.05)。与黄芪糖组相比,仅ESP和FSP中剂量组对K562和FSP中剂量组对YAC-1细胞增殖活性的抑制作用显著高于黄芪糖组(P<0.05)。
FCM结果显示,ESP和FSP均能引起细胞周期时相的改变,阻滞S180细胞于G1期,诱导细胞凋亡,并以中剂量组的作用效果最为明显(P<0.05)。
Extraction, isolation, purification and structural identification of polysaccharide from Spirulina (Arthrospira) platensis of alkaline lakes in Erdos Plateau and Chad Lake in Africa were studied, and also comparatively studied the mechanism of antitumor, activity of antibacteria and antitumor of the polysaccharide. The aim of this paper is to establish the basis of theoretical and applied research on developing polysaccharide of S. (A.) platensis of alkaline lakes in Erdos Plateau and its products, screening new antitumor drugs, natural alternatives to antibiotics and immunostimulant. The thesis is divided into four parts:
(1)Polysaccharide from S. (A.) platensis of two ecogeographic species were extracted with the traditional water extraction and alcohol precipitation method, and purified preliminarily with the trichloroacetic acid and active carbon method. The two sorts of Polysaccharide were further purified with SephadexG-100 gel column chromatography, ESP of polysaccharide from S. (A.) platensis of alkaline lakes in Erdos Plateau and FSP of polysaccharide from S. (A.) platensis of Chad Lake in Africa were achieved.
ESP and FSP were homogeneous component, which have been proved with Paper chromatography, UV spectrum, specific rotation and SephadexG-100 gel column chromatography methods. ESP was milky white flocculent crystal, FSP was light yellow flocculent crystal after freeze-drying by low temperature, and they were both soluble in water. The Polysaccharide′content of ESP and FSP were 89.75% and 62.87%, which have been determined with Sulfuric acid-phenol method. Non-polysaccharide components were mainly ash and protein in FSP and ESP, which have been verified too. ESP and FSP were polysaccharide combined protein possibly that were conjectured.
The molecular weight and structure of ESP and FSP were studied with SephadexG-150 gel column chromatography, TLC, HPLC, IR and NMR methods on the basis of above studies. The results showed that the molecular weight of ESP and FSP were 77625 Dr and 85114 Dr, the connection pattern of single sugar in ESP were mainly [α-Glc(1→4)-]n, including fewα-Glc(1→6)-,α-Fuc(1→2)-,β-Xyl(1→3), -α-Rha as ended glycosylation, The monosaccharide of constituting ESP′were mainlyα-pyranose, largest in glucose, followed by rhamnose and few in arabinose, xylose, galactose, mannose and fructose. The connection pattern of single sugar in FSP wereα-Glc(1→6)- andα-Xyl(1→2), -β-Xyl, -β-Gal and -α-Rha were ended glycosylation, the monosaccharide of constituting FSP were mainlyα- orβ- type glucopyranose, followed by rhamnose and few xylose, mannose, galactose and arabinose.
(2)The antibacterial function of ESP and FSP were studied in vitro with cup-plate method. The results showed that ESP and FSP had bactericidal activity, but inhibitory effect to bacteria was related to concentration of polysaccharide and species of bacteria. Inhibition of ESP was better significant to Shigella dysenteriae in the concentration of 20 mg/mL and 40 mg/mL, and the diameter of bacteriostasis circle were 14.17 millimeters and 12.16 millimeters. FSP had bactericidal activity to Shigella dysenteriae, Pseudomonas aeruginosa and Proteus vulgaris in the concentration of 20 mg/mL, the diameters of the inhibition zone were 12.13 millimeters, 9.10 millimeters and 9.07 millimeters.
(3)The inhibitory effect of ESP and FSP to S180 ascites tumor in mice has been studied with the animal model of S180 ascites tumor. And then the effect of ESP and FSP on proliferation of spleen lymphocyte cell and the concentration of immune cell factors in serum were detected with MTT and ELISA methods, the molecular mechanism of immune enhancement of ESP and FSP were studied preliminarily to tumor-bearing mice. The results showed that ESP and FSP could improve the survival quality of tumor-bearing mice, inhibit the growth of S180 transplanted in mice, reduce the scope of tumor invasion in the body, lengthening survival time of tumor-bearing mice. Effect of the medium dose of ESP and FSP was most significantly on the survival time of tumor-bearing mice, the lengthening rate of life were 50.67% and 52.00%. ESP and FSP could reduce injury of organ in tumor-bearing mice, raising the thymus and spleen index, thus increasing immune function, enhance the levels of IL-2, TNF-αand IFN-γin serum of tumor-bearing mice when inhibit growth of tumor. ESP and FSP could promote the proliferation of spleen lymphocyte cell induced by ConA In vitro. The influence of medium dose of ESP and FSP to cell factors, the proliferation of spleen lymphocyte and immune organ index were most significantly. The above results show that ESP and FSP of appropriate dose can inhibit S180 ascites tumor significantly, enhance T cell-driven immune function, improve the immune regulate function of body. Immune regulate effect may be one of the mechanisms of antitumor.
(4)The effect of ESP and FSP on growth of sarcoma 180, mouse leukemia cells L1210, human chronic myelogenous leukemia K562 and mouse lymphoma cells YAC-1 had been studied with MTT methods, meanwhile effect of ESP and FSP on cell cycle and cell apoptosis of S180 have been studied too with flow cytometry. The results showed that ESP and FSP of different dose all had inhabitation to different degrees on above tumor cells in vitro. But, only the medium doses of ESP and FSP had a inhibition rate more than 30%, reached 35.88% and 33.98%. The medium dose of ESP and FSP all had the more obviously inhibition to the cell proliferation on above tumor cells, but the inhibition effect were significantly lower than CTX group to the S180, YAC-1 and K562 (P<0.05). Comparison with Astragalus sugar group, only the medium dose of ESP and FSP to K562, the medium dose of FSP to YAC-1 had more significant inhabitation than Astragalus sugar group (P<0.05).
The results of FCM showed that, ESP and FSP could cause cell cycle changes, block S180 cells in the G1 phase, induce apoptosis, and the effect of the medium dose of ESP and FSP were most significant (P<0.05) comparison with each other.
引文
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