羧甲基壳聚糖论文：羧甲基壳聚糖对肿瘤细胞及组织生长影响的研究

羧甲基壳聚糖论文：羧甲基壳聚糖对肿瘤细胞及组织生长影响羧甲基壳聚糖论文：羧甲基壳聚糖对肿瘤细胞及组织生长影响 的研究的研究 【中文摘要】:羧甲基壳聚糖(carboxymethyl chitosan ,CM- CTS)是目前壳聚糖改性研究和应用研究最多的一种壳聚糖衍生物,是 经羧甲基化接枝改性而成,具有优良的水溶性、成膜性、保湿性等, 在日化、食品、医药、生物医用材料等领域中具有较多的研究和应 用。CM-CTS 作为生物医用材料在烧伤敷料、术后防粘连、止血、促 进创伤修复、药物载体、酶抑制剂等方面已有应用,但对 CM-CTS 作 为生物医用材料应用在临床的肿瘤手术切除创面的安全性评价方面 研究报道很少。故在细胞水平及组织水平上开展 CM-CTS 对肿瘤生长 的影响具有重要的理论意义和实际意义。本课题以四种不同分子量、 不同羧化度及不同脱乙酰度的 CM-CTS 为研究对象,开展了 CM-CTS 理 化性质测定、体外细胞毒性评价、体内应用安全性评价及对机体免 疫调节的影响,为 CM-CTS 作为生物医用材料在临床应用的安全性提 供理论依据。方法:实验分四个部分:1、对四种 CM-CTS 材料进行纯 化及理化性质分析,采用 HPLC 法测定其分子量,酸碱电位滴定法测定 其羧化度及脱乙酰度,并对水分及灰分等性能进行了研究。2、在细 胞水平,采用显微观察结合 MTT 法测定四种 CM-CTS 对人正常肝细胞 L02、人肝癌细胞 Bel-7402、人胃癌细胞 SGC-7901 及人宫颈癌细胞 Hela 体外生长的影响。3、初步探讨 CM-CTS 对正常肝细胞及肝癌细 胞生长影响差别可能机制。ELISA 方法检测 CM-CTS 对 L02 细胞及 Bel-7402 细胞作用 24h 时转化生长因子-(TGF-),对 Bel-7402 细胞分泌血管内皮生长因子(VEGF)水平影响。4、考察 CM-CTS 体内 应用安全性。构建小鼠肉瘤 S180 及移植性肝癌 Heps 模型,腹腔注射 分子量为 340kDa 的 CM-CTS 给药,计算抑瘤率,并测定 S180 肉瘤小鼠 免疫器官指数、血清白细胞介素-2(IL-2)及肿瘤坏死因子-(TNF- )水平。结果:1、CM-CTS 纯化至细胞使用级别并对其主要理化参 数进行了测定。2、细胞水平结果显示四种 CM-CTS 在 50800g/ml 浓度范围内对 L02 细胞增殖具有显著促进作用,其相对增值率为 114%126%;而 CM-CTS 在 505000g/ml 浓度范围内,作用 2d, 4d 时,对 Bel-740、SGC-7901 及 Hela 细胞的增殖均具有一定的抑制作用,抑 制率在 5%20%。因此,四种 CM-CTS 作为生物医用材料在细胞水平上 是安全的。3、CM-CTS 在 505000g/ml 浓度范围内能显著提高 L02 细胞 TGF- 分泌水平,而对 Bel-7402 细胞 TGF- 及 VEGF 分泌水平 具有显著抑制作用。这与 CM-CTS 对两种肝细胞生长影响结果趋势相 近。4、实验显示大分子 CM-CTS 非但不能促进实体瘤模型小鼠的肿 瘤增长,反而具一定程度的抑瘤作用。低、中、高剂量 CM-CTS 对 Heps 小鼠抑瘤率分别为:12.9%、18.2%及 22.1%,对 S180 肉瘤生长的 抑制率分别为:29.4%、31.6%及 39.9%。此外,CM-CTS 在一定浓度范 围内能提高 S180 荷瘤小鼠免疫器官指数及外周血请中的 IL-2 及 TNF- 的水平,对机体免疫力有保护和调节作用。结论:分子量不同、 羧化度不同及脱乙酰度不同的四种 CM-CTS 对 L02 细胞体外增殖均具 促进作用,对 Bel-7402、SGC-7901 及 Hela 细胞增殖无促进作用并有 一定的抑制作用,对相关细胞因子分泌水平影响结果与细胞水平结果 相近,对小鼠移植性肝癌 Heps 及 S180 肉瘤具一定抑制作用,并能提 高机体免疫力。因此,CM-CTS 其作为生物医用材料在机体中应用是 安全的。 【英文摘要】Purpose: Carboxymethyl chitosan (CM-CTS) is a water soluble chitosan derivative by introducingCOOH groups to chitosan molecule and endows it some outstanding biological properties. CM-CTS has been a research focus in the field of pharmaceutical and biomedical industry for its unique physicochemical and biological properties such as biocompatibility, biodegradability, degradation controllability and antimicrobial activity. There have also been some reports on the therapeutic utilities of the CM- CTS in the fields of antimicrobes, hemostatic, wound healing, reducing the formation of post surgical adhesions, drug carrying, enzyme inhibitor and so on. As versatile biomedical material, CM-CTS always conjugates or entraps or self-assembles with agent and extensively applies in tumor therapy research. However, safety evaluation of CM-CTS has been focused on the compounds and in vitro model, little reported work of CM-CTS perse safety both in vitro and vivo has been discussed, especially in tumor therapy. Moreover, the actual metabolic pathway and degradation products of CM-CTS in the body have been as yet unknown. Therefore, additional biological safety research of CM-CTS in application is necessary.In this study, the biological safety of CM-CTS with different molecular weights (MW), different substituted degrees (DS) and different degree of deacetylation (DD) on clinical application is investigated both in vitro and in vivo. CM-CTS is purified, characterized and its cytotoxicity is investigated by using normal cell and three tumor cells in vitro, and the influence of CM-CTS on the growth of sarcoma 180 and Heps tumor and immune response of tumor-bearing mice are also investigated, providing experimental basis for CM-CTS utilization in tumor application as biomedical material. Methods:1. Four CM-CTS samples with different MW, different DS and different DD are purified and their mainly physicochemical properties are measured. The MW of CM-CTS are measured with gel permeation chromatography using HPLC; DS and DD of CM-CTS are determined by potentiometric; The moisture and ash properties of CM-CTS are also determined.2. Human normal liver cell L02, human hepatoma cell Bel-7402, human gastric cancer cell SGC-7901 and human cervical carcinoma cell Hela are used to evaluate the cytotoxicity effects of CM-CTS by MTT assay in vitro and the morphology of cell is observed by inverted microscope.3. The levels of human transforming growth factor-(TGF-) and human vascular endothelial growth factor (VEGF) secreted by L02 and Bel-7402 cells are measured by ELISA.4. In vivo, the transplant tumor models of Heps and sarcoma 180 are established in mice and 340kDa CM-CTS is administered through intraperitoneal injection to investigate tumor inhibitory effect. The tumors are weighed to calculate the tumor inhibition rate. Immune organs including spleen and thymus of the mice are also removed and weighed to obtain the index of the spleen and thymus. Serum IL-2 and TNF- level of sarcoma-180-bearing mice are also investigated.Results:1. Four CM-CTS samples with different MW, different DS and different DD are purified and their main chemical and physical parameters are measured.2. Four CM-CTS samples with different different MW, different DS and different DD could promote the proliferation of L02 cells within the concentration of 50800g/ml, but not promote even inhibit the proliferation of Bel-7402, SGC- 7901 and Hela cells in certain concentration ranges and incubation time. The RGR value are 114%126% of L02 cells but 80%95% for that of three tumor cell lines: Bel-7402, SGC-7901 and Hela cells.3. Four CM-CTS samples with different MW, different DS and different DD also could improve the TGF-secretion of L02 cells, whereas decrease that of Bel-7402 cells. The level of VEGF secreted by Bel- 7402 cells is also inhibited by CM-CTS to some extent.4. CM-CTS exhibits anticancer effect in a dose-dependent manner. The tumor inhibition rates of low, medium and high doses of CM-CTS are 12.9 %, 18.2 % and 22.1 % to the growth of Heps tumor and 29.4 %, 31.6 % and 39.9 % to the growth of sarcoma 180 tumors, respectively. CM-CTS also could enhance body immune function in the certain dose via elevation of serum IL-2 and TNF-level and the spleen index and thymus index in treated mice.Conclusion: CM-CTS with different MW, different DS and different DD are safe to cell and exhibit twofold bioactivities in the culture of human normal liver cell L02 and three tumor cells: Bel-7402, SGC-7901 and Hela. Results at cytokine levels are compatible with the observations at liver cell levels. In vivo, CM-CTS are nontoxic to body and could enhance body immune function and exhibited anticancer effect to some extent and these results strongly suggest that CM-CTS is safe in tumor application as biomedical material. 【关键词】羧甲基壳聚糖 肿瘤 生物材料 细胞因子 【英文关键词】Carboxymethyl chitosan Tumor Biomaterial Cytokine 【备注】索购全文在线加我：1.3.99.3.8848 同时提供论文一对一写作指导和论文发表委托服务 【目录】羧甲基壳聚糖对肿瘤细胞及组织生长影响的研究 摘要 5-7 Abstract 7-9 第一章 文献综 述 15-32 1 壳聚糖概述 15-18 1.1 壳 聚糖结构性质 15-16 1.2 壳聚糖的物理改性 16 1.3 壳聚糖的化学改性 16-18 1.3.1 酰化改性 16 1.3.2 烷基化改性 16-17 1.3.3 醚化改性 17 1.3.4 酯化改性 17 1.3.5 希夫碱反应 17 1.3.6 季铵化 17 1.3.7 氧化改性 17-18 1.3.8 交联改性 18 1.3.9 接枝共聚改性 18 2 羧甲基壳聚糖概述 18-23 2.1 羧甲基壳聚糖结构 18-19 2.2 羧甲基壳聚糖性质 19-20 2.3 羧甲基壳聚糖的应用现状与前景展望 20-23 2.3.1 在 医药方面的应用 20-22 2.3.2 在生活日用品方面的应 用 22 2.3.3 在农业上的应用 22-23 2.3.4 在环保方面的应用 23 3 肿瘤疾病概述 23- 26 3.1 肿瘤发生机理 23-24 3.2 肿瘤疾病 的研究进展 24-25 3.3 肿瘤细胞与正常细胞主要差别 25-26 4 细胞因子 26-31 4.1 细胞因子分 类 26-29 4.1.1 白细胞介素（IL) 26 4.1.2 集落刺激因子（CSF) 26-27 4.1.3 干扰素 （IFN) 27 4.1.4 肿瘤坏死因子（TNF) 27 4.1.5 转化生长因子家族（TGF) 27 4.1.6 趋化因子 家族（chemokinefamily) 27-28 4.1.7 其它生长因子 28-29 4.2 细胞因子在肿瘤研究中的意义 29-31 4.2.1 VEGF 在肿瘤研究中的意义 29-30 4.2.2 TGF- 在肿瘤研究中的意义 30 4.2.3 IL-2 在肿瘤研究 中的意义 30 4.2.4 TNF- 在肿瘤研究中的意义 30-31 5 立题依据 31-32 第二章 四种 CM- CTS 材料纯化及理化性质测定 32-38 1 材料、试剂与 仪器 32-33 1.1 材料与试剂 32 1.2 主 要仪器 32-33 2 实验方法 33-36 2.1 材料的制备与纯化 33-34 2.2 材料性质的测定 34-36 2.2.1 CM-CTS 水分测定 34 2.2.2 CM-CTS 灰分测定 34 2.2.3 CM-CTS 分子量的测定 34-35 2.2.4 CM-CTS 脱乙酰度的测定 35 2.2.5 CM-CTS 羧基取代度的测定 35-36 3 结果与讨 论 36-38 第三章 四种 CM-CTS 对正常细胞及肿瘤细 胞生长的影响 38-58 1 材料、试剂与仪器 38-40 1.1 材料与试剂 38-39 1.2 溶液配制 39 1.3 主要仪器 39-40 2 实验方法 40-41 2.1 四种细胞培养活性鉴定 40 2.2 CM-CTS 对四种细 胞生长影响 40 2.3 数据分析 40-41 3 结果与讨论 41-57 3.1 四种 CM-CTS 对正常肝细胞 L02 细胞生长的影响 41-45 3.1.1 四种 CM-CTS 对正 常肝细胞 L02 细胞形态的影响 41-44 3.1.2 四种 CM- CTS 对正常肝细胞 L02 细胞活性的影响 44-45 3.2 四种 CM-CTS 对肝癌细胞 Bel-7402 细胞生长的影响 45-49 3.2.1 四种 CM-CTS 对肝癌细胞 Bel-7402 细胞形态的影响 45- 48 3.2.2 四种 CM-CTS 对肝癌细胞 Bel-7402 细胞活性的 影响 48-49 3.3 四种 CM-CTS 对胃癌细胞 SGC-7901 细胞活性的影响 49-53 3.3.1 四种 CM-CTS 对胃癌细 胞 SGC-7901 细胞形态的影响 49-52 3.3.2 四种 CM- CTS 对胃癌细胞 SGC-7901 细胞活性的影响 52-53 3.4 四种 CM-CTS 对宫颈癌细胞 Hela 细胞生长的影响 53-57 3.4.1 四种 CM-CTS 对宫颈癌细胞 Hela 细胞形态的影响