【作者】 刘少芳；

【导师】 陈靠山；

【作者基本信息】 山东大学 ， 生化与分子生物学， 2007， 硕士

【摘要】 菊糖和果寡糖在实验中发现具有各种免疫活性和抗肿瘤作用。菊科植物如菊苣(chicory)和菊芋(Jerusalem artichoke)是目前工业化生产菊糖和果寡糖的主要来源。另一种重要的菊科植物牛蒡，其根部富含菊糖。本实验室已从其根部分离纯化得到一种菊糖构型的低聚果糖，命名为牛蒡低聚果糖(Burdock fructooligosaccharide，BFO)。BFO与其它菊科植物来源的菊糖和果寡糖具有相似的化学结构。初步实验证实BFO能促进植物的生长，是一种植物抗病诱导子。同时体外实验证明BFO能促进双歧杆菌的生长及小鼠脾细胞的增殖，提示BFO具有益生素特征和作为免疫刺激剂。目前有关BFO对机体的免疫活性的报道很少。因此对BFO的免疫调节作用的研究，将会为其进一步开发和应用提供科学的依据。本研究首先探讨一种简便高效的去除BFO中细菌内毒素的方法，以确保其在体内外活性测定中结果的可靠性。然后考察无内毒素BFO体外给药对主要免疫细胞的直接作用，进而对正常小鼠、荷瘤小鼠以及环磷酰胺致免疫低下小鼠的免疫功能进行了研究，并初步探讨了BFO的抗肿瘤活性。1、牛蒡低聚果糖细菌内毒素的去除内毒素是大多数生物材料的污染源，它的存在使得许多生物、药物实验出现混乱的结果，给生产带来许多困难。本实验采用DEAE-纤维素去除BFO中的细菌内毒素，内毒素含量降低了约25倍，回收率可达70％，证明是一种较合适的去除BFO所污染内毒素的方法。2、牛蒡低聚果糖体外给药对免疫细胞功能的影响本实验研究了BFO体外给药对小鼠脾淋巴细胞和腹腔巨噬细胞活性的影响。淋巴细胞在免疫调控中起重要作用，其增殖反应是反映淋巴细胞活化和功能的重要指标。实验结果表明，BFO(25-1000μg／ml)作用能提高小鼠脾淋巴细胞的增殖活性，呈剂量依赖性，在浓度为1000μg／ml时增殖活性达到最大。说明BFO在一定浓度范围内具有刺激淋巴细胞增殖的有丝分裂源活性。同时，BFO在一定浓度范围内(25-1000μg／ml)刺激经ConA作用的小鼠淋巴细胞增殖，二者具有协同作用。提示BFO可增强T细胞介导的细胞免疫功能。巨噬细胞是机体免疫系统的一种重要的免疫细胞。巨噬细胞在机体的免疫防御中起重要作用，如病原的吞噬作用、细胞因子的产生以及外来抗原的水解处理和递呈。NO是一种具有多种生理功能的信息分子及效应分子，在巨噬细胞发挥细胞毒作用、免疫调节作用及细胞内杀菌作用等方面具有重要意义。BFO在体外单独作用不能刺激经硫代乙醇酸盐培养基活化的小鼠腹腔巨噬细胞产生NO。BFO在不同的浓度范围内对LPS刺激的巨噬细胞产生NO的作用不同。在浓度为6.25-100μg／ml范围时，随着BFO浓度的增加，抑制了NO的产生，呈剂量依赖性。在浓度为500-1000μg／ml时，随着浓度的升高，反而明显刺激NO的产生，在1000μg／ml时达到极显著差异水平(P＜0.01)。提示BFO在不同的浓度条件下对巨噬细胞产生NO的作用机制不同。酸性磷酸酶是活化的巨噬细胞产生的一种溶酶体酶，也反应了巨噬细胞的吞噬功能。实验显示BFO能明显促进巨噬细胞酸性磷酸酶的活性。在浓度为25μg／ml时活性开始增强，随着浓度的增加，作用逐步增强，在浓度为50-1000μg／ml的范围内呈剂量依赖性。与对照组相比，在100pg／ml时即达到极显著差异(P＜0.01)。BFO对淋巴细胞的增殖作用和对巨噬细胞功能的刺激作用，说明其可能作为一种潜在的生物反应调节剂(Biological response modifier，BRM)在机体的免疫反应中起作用，可能通过刺激淋巴细胞和巨噬细胞的作用来帮助整个免疫系统活化。3、牛蒡低聚果糖体内给药对小鼠的免疫调控及抗肿瘤作用本实验分别研究了BFO灌胃给药对正常小鼠、荷瘤小鼠以及环磷酰胺致免疫低下小鼠的免疫功能的影响。通过对巨噬细胞的吞噬活性及细胞因子的产生、脾细胞分泌抗体及脾淋巴细胞增殖活性的测定，来探讨BFO对机体的细胞免疫和体液免疫功能的影响。BFO(100、250、500和1000mg／kg／day)灌胃给药能提高正常小鼠巨噬细胞NO的产生量、增强巨噬细胞的吞噬及酸性磷酸酶活性，而对正常小鼠的B细胞和血清介导的溶血反应及脾淋巴细胞的增殖无明显影响。BFO给药组(250、500和1000mg／kg／day)能提高免疫低下小鼠巨噬细胞酸性磷酸酶活性和吞噬中性红的能力，同时还能提高脾淋巴细胞的增殖活性以及显著增强小鼠B细胞和血清介导的溶血反应，说明BFO能对抗环磷酰胺诱发的免疫抑制作用。BFO对荷瘤小鼠的免疫刺激作用通过检测荷瘤小鼠巨噬细胞的吞噬活性和NO产生量来评价，与荷瘤小鼠对照组相比，BFO给药组(250、500和1000mg／kg／day)能明显提高小鼠巨噬细胞吞噬中性红的能力和增加产生NO的量，表明BFO体内给药能增强荷瘤小鼠巨噬细胞的吞噬功能和产生NO的能力。另外，本实验还研究了BFO灌胃给药对小鼠S180肉瘤的抑制作用。实验结果表明，BFO接瘤后给药，低剂量组(250和500 mg／kg／day)无明显的抑瘤作用，高剂量组(1000mg／kg／day)初步显示出抗肿瘤活性，抑瘤率为19.01％。预给药7天后接瘤，不同剂量组的抑瘤率均明显提高，低剂量组(250和500mg／kg／day)的抑瘤率为24.26和25.53％，而高剂量组达到33.14％。以上研究结果表明，BFO具有明显的免疫增强作用及抗肿瘤活性，其作用机制可能与促进淋巴细胞增殖及增强巨噬细胞的活性有关，从而产生显著的免疫调节作用和BRM活性。这一结果为应用BFO防治肿瘤等免疫缺陷性疾病莫定了理论基础，并为其进一步开发为药品提供了实验依据。更多还原

【Abstract】 Inulin and oligofructose are known to modulate immune functions and exert anti-tumor activities. The main sources of inulin and oligofructose that are used in the food industry are chicory and Jerusalem artichoke. As one of the immportant Compositae family plant, Burdock (Arctium lappa L, var. Herkules)’s roots are also abundant in inulin. A low-molecular-weight inulin-type oligosaccharide named as Burdock fructooligosaccharide (BFO) has been obtained from the roots and its structure characteristics have also been identified in our lab. BFO has a chemical structure similar to that of polyfructosides from the plants of Compositae family. Preliminary studies have shown that it can act as an inducer in the plant growth and promote the increase amount of bifidobacteria and the proliferation of murine splenocytes in vitro. These studies suggest that BFO are prebiotics and immuno-stimulating agents. However, the immunomodulatory effects of BFO are rarely documented. Therefore, to understand its immunomodulatory effects will contribute to its further reseach and developmemt.In the present study, we first probed into a low-cost method to exclude the endotoxin contamination. Then the immunomodulatory effects of BFO on mice were investigated in vitro and in vivo. The anti-tumor activities of BFO on S180 mice were also studied.The exclusion and determination of endotoxin contamination in Burdock fructooligosaccharideGram-negative bacterial LPS (i.e., endotoxin) is a main contaminant in many biochemical materials prepations which exhibit a potential interference by its innate stimulatory properties. In our study, DEAE-cellulose was used to exclude the LPS-like contaminants in BFO. We demonstrated that DEAE-cellulose was a suitable method to exclude endotoxin contaminant in BFO, with the LPS-content decreasingas low as 25 folds and the recovery rate reaching 70%.Effects of BFO on in vitro immunomodulatory activity of mouse immune systemImmunomodulatory activity of BFO was examined on the proliferation response of murine splenocytes and the macrophages in vitro. Lymphocytes play a pivotal role in the regulation of immune responses. And its rapid proliferation is crucial in the assessment of lymphocyte functions. Our study observed that treatment with 25-1000 μg/ml BFO enhance the proliferation of splenocytes in a dose-dependent manner, and the maximal activity was obtained at the concentration of 1000 μg/ml. Also, 25-1000 μg/ml BFO dose-dependently enhance ConA-induced splenocyte proliferation. It was suggested that BFO has a direct effect on T cell function and might influence T cell-induced cellular immunity.Macrophages are important cells which play key roles in immune system defense, including the phagocytosis of pathogens, the production of many cytokines, and the proteolytic processing and presentation of foreign antigens. Nitric oxide (NO) produced during macrophage activation is a new type of effector molecular which involved in many physiological and pathological processes such as nonspecific host defense, antimicrobial and anti-tumor activities and cytotoxic effcet. The results showed that BFO had no direct effect on the production of NO in murine peritoneal macrophages. Different effects were obtained when BFO treated with LPS. BFO exhibited inhibitory effects in low concentration while the contrary effcets in high level.Acid phosphatase is one kind of lysosomal enzyme produced by activated macrophages which is also an important indicate in the assessment of macrophage function. BFO can significantly increase the acid phosphatase activity at a concentration of 50-1000 μg/ml in a dose-dependent fashion. BFO (100 μg/ml) can markedly promote the acid phosphatase activity compared to a control (p<0.01). The immunomodulatory in vivo and anti-tumor activity of Burdock fructooligosaccharideThe modulation of BFO on the immune system in normal mice, cyclophosphamide (CTX) -induced immunosuppressive mice and S180-bearing mice was investigated. The phagocytosis, NO production and acid phosphatase activities in macrophage, the level of serum hemolysin, B cell mediated haemolytic effect and lymphocyte proliferation were tested to investigate the cellular and humoral immunity in animal models.The mice were treated p.o. with BFO (100, 250,500 and 1000 mg/kg/day) for 10 days. Our study showed that BFO could significantly increase the phagocytosis, the release of NO and the acid phosphatase activity in macrophage, while the level of serum hemolysin, B cell mediated haemolytic effect and spleen lymphocyte proliferation activity were unchanged in normal mice. However, naearly all the immune parameters mentioned above were improved in CTX-induced immunosuppressive mice, suggesting that BFO can antagonize the immuosuppression induced by CTX. We also found that BFO can significantly intensify the macrophage phagocytosis and the NO production in S180-bearing mice. These results suggest that BFO has a highly significant effect on the improvement of immune system in normal, immunosuppressive and tumor-bearing mice.Then, we investigated the antitumor effects of BFO in S180-bearing mice. BFO inhibited the growth of S180 tumor only at a high dose of 1000 mg/kg/day when treated after tumor inoculation, with inhibitory rate 19.01%. In contrast, when administrated to the mice for 7 days before tumor inoculation at a dose of 250, 500 and 1000 mg/kg/day, BFO could significant exhibt antitumor activity. The inhibitory rate was 24.26%, 25.53% and 33.14%, respectively.The results demonstrated that BFO exerts strong immunomodulatory and anti-tumor effects on experimental mice which may involve in the augmentation of lymphocyte and macrophage activities. Our research may make contribution to the theoretic basis for the development of BFO as a new Biological response modifier (BRM).更多还原