湖北理工学院 毕业设计（论文）
摘 要
目的：地木耳(Nostoc commune vauch)是属于藻类蓝藻纲念珠藻科(Nostocaceae)，
全球广泛分布，资源十分丰富。已有研究表明地木耳含有丰富的氨基酸、糖苷、维生素、钙、磷等，在抑菌、抗氧等方面具有很高的活性。而近些年来，有很多关于多种植物、真菌多糖抗肿瘤领域的研究。随着地木耳以药材的身份逐渐走入了研究人员的视野，地木耳多糖的药理药效也开始得到重视。本课题拟针对地木耳多糖的抗肿瘤活性开展研究。
宫颈癌是女性患者常见的恶性肿瘤之一，原位癌高发的年龄为30～35
岁，浸润癌为45～55岁，而且近年来其发病更有向年轻女性发展的趋势，已是女性患者的一大杀手，而肿瘤细胞的侵袭和转移是导致放疗、化疗失败的主要因素。本实验利用多糖组和对照组宫颈癌细胞培养，并通过划痕实验，确定地木耳多糖对宫颈癌细胞侵袭的作用效果。
方法：首先，用水醇法按照60：1液料比，温度控制在90℃，反复提取三次，合
并提取液抽滤，并进行浓缩。加入无水乙醇，低温过夜。离心，取沉淀，并用savege试剂去除蛋白，干燥成粉末。然后绘制葡萄糖标准曲线，测出提取多糖的含量。将地木耳多糖配制成三种不同浓度溶液，加入宫颈癌HeLa细胞培养皿中，进行划痕实验，培养0、6、12、24、48小时后观察划痕的修复。最后进行统计学处理。
结果: 本实验利用水提法对地木耳进行提取，提取率为7.86%。地木耳多糖有抑制
宫颈癌Hela细胞侵袭的作用，在三种浓度中，40μg/ml的抑制效果强，并且具有剂量依赖性。
结论：地木耳多糖可抑制宫颈癌Hela细胞的侵袭能力，有望用于宫颈癌的治疗。 关键词：地木耳 多糖 宫颈癌 侵袭
湖北理工学院 毕业设计（论文）
Abstract
Objective：Nostoc commune Vauch commonly known as Nostoc commune, Nostoc commune is a global wide species, mainly distributed in Karst karst area and limestone karstzone in Karst district. Nostoc contains a lot of amino acid, glucoside, vitamin, calcium, phosphorus. In recent years, there are many researches on polysaccharides efficacy in this direction. With the fungus medicine identity gradually into there search field of vision, pharmacological effect of polysaccharides of Nostoc commune also get attention.
Cervical cancer is one of the most common malignant tumor of female patients, in some cancer at the age of 30 to 35 years of age, invasive carcinoma was 45 ~ 55 years old, and more in recent years the incidence to the development trend of young women. Tumor invasion and metastasis is one of the main lethal factor. This experiment using polysaccharide group and control group to determine the effect on cultured cervical cancer cells.
Methods: Firstly, water alcohol method according to the 60:1 ratio of liquid to solid, temperature control in 90 ℃, repeated extraction. Repeated three times of extraction, merging the extracted liquid filtration, and concentrating. Adding anhydrous ethanol, cold night. Centrifugal, and precipitation, and Savege reagent was used to remove protein, drying into powder. Secondly, draw the standard curve of glucose, measure the content of polysaccharides extracted from. The fungus polysaccharide preparation of three different concentration solutions, adding to cervical cancer HeLa cell culture dishes. For wounding healing test, HeLa cell cultured for 0, 6, 12, 24, 48 hours. Finally, statistical analysis.
Results: To the Nostoc polysaccharide solution inhibited the invasive cervical carcinoma Hela cells, and 40μg/ml of the strongest inhibitory effect of the Nostoc polysaccharide.
Conclusion: after the processing, compared with the control group, the experimental findings, Nostoc polysaccharide solution, reduce the ability of cells to repair the scratches, as shown in figure shows more of Nostoc polysaccharide suppresses the motion ability of cervical cancer cell line. Key words: Nostoc commune polysaccharide cervical cancer invade
湖北理工学院 毕业设计（论文）
目录
1 问题的提出 .......................................................................................................................... 1
1.1 研究的背景 .......................................................................................................................... 1 1.2 研究的目的和意义 ............................................................................................................... 1
2 文献综述 ................................................................................................................................ 2
2.1 地木耳的研究进展 ............................................................................................................... 2 2.1.1地木耳的性状.................................................................................................................... 2 2.1.2 地木耳的营养成分 ........................................................................................................... 2 2.1.2.1 蛋白类 .......................................................................................................................... 2 2.1.2.2 脂类 .............................................................................................................................. 3 2.1.2.3 氨基酸类....................................................................................................................... 3 2.1.2.4 多糖类 .......................................................................................................................... 3 2.1.2.5 维生素类....................................................................................................................... 4 2.1.2.6 矿物质类....................................................................................................................... 4 2.1.2.7 色素类 .......................................................................................................................... 4
2.1.3 医药领域 .......................................................................................................................... 5 2.1.3.1 地木耳在医药中的功效 ............................................................................................... 5 2.1.3.2 地木耳的药理作用研究进展 ....................................................................................... 6 2.1.4 食品领域 .......................................................................................................................... 7 2.1.5 保护环境 .......................................................................................................................... 8 2.1.6 农林牧业 .......................................................................................................................... 8
2.2肿瘤侵袭机制........................................................................................................................ 8 2.2.1 肿瘤 .................................................................................................................................. 9
2.3天然抗肿瘤活性物质研究现状 .......................................................................................... 10 2.4 划痕实验 ............................................................................................................................ 11
3 研究方法 .............................................................................................................................. 13
3.1 材料与仪器 ........................................................................................................................ 13 3.1.1 材料 ................................................................................................................................ 13 3.1.2 仪器 ................................................................................................................................ 13
3.2 地木耳多糖的提取 ............................................................................................................. 13 3.2.1 绘制标准曲线................................................................................................................. 13 3.2.2 地木耳的提取................................................................................................................. 14 3.2.3 Sevage试剂除蛋白纯化及浓缩 .................................................................................... 14 3.2.4 地木耳多糖提取率及其含量的测定 ............................................................................. 14
3.3 多糖溶液的配制................................................................................................................. 14 3.4 HeLa细胞分板培养及划痕实验 ........................................................................................ 14 3.4.1 试剂及设备..................................................................................................................... 14 3.4.2 细胞培养 ........................................................................................................................ 15
湖北理工学院 毕业设计（论文）
3.4.3 地木耳多糖抑制Hela细胞侵袭活性检测 ................................................................... 22 3.5 实验结果 ................................................................................................................................. 23 3.5.1地木耳多糖含量测定 ...................................................................................................... 23 3.5.2 抑制Hela细胞侵袭活性检测 ....................................................................................... 24
4 结论和建议 ........................................................................................................................ 27 参考文献 .................................................................................................................................... 28 致 谢 ............................................................................................................................................. 29
湖北理工学院 毕业设计（论文）
1 问题的提出
1.1 研究的背景
肿瘤侵袭扩散是恶性肿瘤的主要特征之一。具有浸润性生长的恶性肿瘤，不仅能在原发部位迅速生长，然后直接蔓延。而且肿瘤还可以通过侵袭等各种途径扩散到身体其他部位（转移），从而导致无法彻底的根治。肿瘤的侵袭扩散能力是肿瘤无法彻底根治的关键。
目前国内和国外对地木耳（Nostoc commune）的化学成分、生物活性和开发利用等研究正处于开始阶段, 对地木耳营养成分进行了一些初步的研究,但是研究结果差别相对较大，不宜采用;对地木耳的化学成分并未进行系统研究,其活性成分的构成及生物活性还有待深入研究;有关化学成分的纯化分离、结构的分析鉴定、活性检测等研究同样不够;针对地木耳功能食品、药品和环保产品的研究开发不够,其资源的综合利用有待进一步发现和探索。
1.2 研究的目的和意义
本课题“地木耳多糖抑制人宫颈癌侵袭能力实验研究”是研究Nostoc commune当中的多糖成分可能具有抑制宫颈癌侵袭的能力，对阻止癌细胞侵犯临近组织和细胞进而转移扩散全身具有重要的意义。原材料地木耳产地遍布多省，资源丰富，容易采集获得。而近些年来，有很多关于多种植物、真菌多糖抗肿瘤领域的研究。本课题研究地木耳多糖抑制人宫颈癌细胞侵袭能力，为抗癌药品的研发和推广、临床及应用提供新的思路。