一种多聚糖类抗癌材料及其制备方法与应用 技术领域 Polysaccharide anticancer material, preparation method and application thereof
本发明涉及一种以多聚糖为基础原料的抗癌材料及其制备方法。 背景技术 The invention relates to an anticancer material based on polysaccharides and a preparation method thereof. Background technique
多聚糖是天然的高分子生物多聚体, 其降解产物为具有弱碱性的氨基 葡萄糖, 对人体组织无毒、 无害。 它除具有优异生物相溶性外, 还具有独 特的生物活性, 能杀灭肿瘤细胞, 并有较强的抑菌活性, 能调节动物体内 胆固醇与脂质代谢。 多聚糖微球、 珠状多聚糖已被研究应用于抗癌药物与 蛋白质的控释***。 制备珠状多聚糖的方法有水油两相法、 乳滴技术、 喷 雾千燥法等, 通常这些制备过程复杂且需要使用有机溶剂与表面活性剂, 由此制备的多聚糖微球不适合物化性质表征观察。 Polysaccharides are natural high-molecular biopolymers, and the degradation products are glucosamine with weak alkalinity, which is non-toxic and harmless to human tissues. In addition to its excellent biocompatibility, it also has unique biological activity, can kill tumor cells, and has strong antibacterial activity, which can regulate cholesterol and lipid metabolism in animals. Polysaccharide microspheres and bead-like polysaccharides have been studied in anti-cancer drugs and controlled release systems of proteins. Methods for preparing bead-like polysaccharides include water-oil two-phase method, emulsion droplet technology, spray drying method, etc. Generally, these preparation processes are complicated and require the use of organic solvents and surfactants. Suitable for observation of physical and chemical properties.
多聚糖具有增强抗肿瘤药物作用, 能促进白细胞介素 2(IL-2)的生成, IL-2对 NK细胞、 T细胞、 B细胞活性均有增强作用。 多聚糖是一种带正 电荷天然高分子生物聚合物, 显示极强的生理活性。 肿瘤细胞表面带有更 高负电荷, 聚阳离子电解质能吸附到癌细胞表面使电荷中和, 抑制肿瘤细 胞生长与转移。 在酸性环境中能形成阳离子基团, 与体细胞产生亲和性, 活化体细胞, 增强机体免疫力 (Hiroshi Ueno,et.al Topical formulations and wound healing applications of chitosan. Advanced Drug Delivery Reviews, 2001, 52:105-115)。多聚糖具有清除体内自由基细胞毒素的能力, 可作为早 期癌症的治疗药物。 Polysaccharides have the effect of enhancing anti-tumor drugs, can promote the production of interleukin 2 (IL-2), and IL-2 can enhance the activity of NK cells, T cells, and B cells. Polysaccharide is a kind of positively-charged natural high-molecular biopolymer, showing extremely strong physiological activity. The surface of tumor cells has a higher negative charge. Polycation electrolyte can adsorb to the surface of cancer cells to neutralize the charge and inhibit the growth and metastasis of tumor cells. Can form cationic groups in acidic environment, produce affinity with somatic cells, activate somatic cells, and enhance body immunity (Hiroshi Ueno, et.al Topical formulations and wound healing applications of chitosan. Advanced Drug Delivery Reviews, 2001, 52 : 105-115). Polysaccharides have the ability to remove free radical cytotoxins from the body and can be used as a treatment for early cancer.
癌细胞会释放大量的癌毒素, 低聚多聚糖在肠道内形成的小分子容易 被肠吸收, 抑制癌毒素在体内的释放, 同时多聚糖吸附在血管壁细胞的表 面, 可抑制癌细胞的转移。 Cancer cells release a large amount of cancer toxins. The small molecules formed by oligosaccharides in the intestine are easily absorbed by the intestine, which inhibits the release of cancer toxins in the body. At the same time, the polysaccharides are adsorbed on the surface of blood vessel wall cells, which can inhibit cancer cells. The transfer.
已有研究报道锌、 钴等金属离子具有较高的抗癌活性。 通过将此类金 属离子负载于具有生物相溶性可降解的多聚糖制备复合抗癌材料, 能够提 高其抗癌活性, 并能发挥缓释、 长效、 无残留作用。 发明内容 Studies have reported that metal ions such as zinc and cobalt have higher anticancer activity. By preparing such metal ions on a biocompatible and degradable polysaccharide to prepare a composite anti-cancer material, the anti-cancer activity can be improved, and the sustained-release, long-acting, and no-residue effects can be exerted. Summary of the invention
本发明的目的是提供一种可作为抗癌药物应用的多聚糖类抗癌材料
及其制备方法。 The object of the present invention is to provide a polysaccharide anticancer material which can be used as an anticancer drug. And its preparation method.
为实现上述发明目的, 本发明一方面提供了一种多聚糖类抗癌材料, 所述多聚糖类抗癌材料是分子量为 10〜90kDa的多聚糖微粒子。 本发明的 多聚糖类抗癌材料还可以是载金属离子的多聚糖微粒子, 其中多聚糖的分 子量为 10〜90kDa, 金属离子含量为 1〜20% (重量)。 In order to achieve the above-mentioned object of the present invention, one aspect of the present invention provides a polysaccharide anticancer material. The polysaccharide anticancer material is a polysaccharide fine particle having a molecular weight of 10 to 90 kDa. The polysaccharide-based anticancer material of the present invention may also be metal ion-containing polysaccharide microparticles, wherein the molecular weight of the polysaccharide is 10 to 90 kDa and the metal ion content is 1 to 20% by weight.
本发明另一方面涉及制备上述多聚糖类抗癌材料的方法, 该方法包 括,在 20〜60°C搅拌下,于稀酸溶液中加入一种或多种分子量为 10〜90kDa 的多聚糖, 配置 0. 1%〜5% (质量百分比)多聚糖酸溶液, 调节溶液 pH值为 6-9, 形成微粒子乳液, 分离所述微粒子并干燥, 即得所述多聚糖类抗癌 材料。 Another aspect of the present invention relates to a method for preparing the above-mentioned polysaccharide anticancer material. The method includes adding one or more polymers having a molecular weight of 10 to 90 kDa to a dilute acid solution under stirring at 20 to 60 ° C. Sugar, configured with a 0.1% to 5% (mass percent) polysaccharide acid solution, adjusting the solution pH to 6-9, to form a microemulsion, separating the microparticles and drying to obtain the polysaccharide anticancer material.
在多聚糖类抗癌材料是载金属离子的多聚糖微粒子的情况下, 该方法 还包括将分离出的所述微粒子洗至中性, 然后加入浓度为 100〜1000mg/L 金属离子溶液中, 振荡吸附并烘干, 得到所需多聚糖类抗癌材料。 In the case where the polysaccharide anticancer material is metal ion-carrying polysaccharide microparticles, the method further includes washing the separated microparticles to neutrality, and then adding the metal ion solution at a concentration of 100 to 1000 mg / L. The polysaccharide-type anticancer material is obtained by shaking absorption and drying.
具体而言, 本发明的多聚糖类抗癌材料有两种制备方法。 Specifically, there are two methods for preparing the polysaccharide anticancer material of the present invention.
方法 1, 制备步骤如下: Method 1, the preparation steps are as follows:
在 20〜60°C搅拌下, 于稀酸溶液中加入分子量为 10〜90kDa的多聚 糖, 配置 0.1%〜5%多聚糖酸溶液, 调节溶液 pH值为 6〜9, 形成微粒子 乳液, 将微粒子乳液沉淀分离, 沉淀物蒸馏水洗涤至中性, 干燥, 即得多 聚糖类抗癌材料。 Add a polysaccharide with a molecular weight of 10 to 90 kDa to a dilute acid solution under stirring at 20 to 60 ° C, configure a 0.1% to 5% polysaccharide acid solution, adjust the solution pH to 6 to 9, and form a microemulsion. The microemulsion is precipitated and separated, and the precipitate is washed with distilled water to neutrality and dried, that is, polysaccharide anticancer material.
方法 2, 制备步骤如下- 在 20〜60°C搅拌下, 于稀酸溶液中加入分子量为 10〜90kDa的多聚 糖, 配置 0.1%〜5%多聚糖酸溶液, 调节溶液 pH值为 6〜9, 形成微粒子 乳液, 沉淀分离, 沉淀物蒸馏水漂洗至中性, 干燥得多聚糖微粒子, 将多 聚糖微粒子干粉置于浓度为 100〜1000mg/L金属离子溶液中, 振荡吸附, 将吸附后的微粒子置于烘箱中烘干, 即得多聚糖类抗癌材料。 Method 2, the preparation steps are as follows-under stirring at 20 ~ 60 ° C, polysaccharide having a molecular weight of 10 ~ 90kDa is added to the dilute acid solution, and a 0.1% ~ 5% polysaccharide acid solution is configured, and the pH of the solution is adjusted to 6 ~ 9, Form a microemulsion, separate the precipitate, rinse the precipitate with distilled water to neutral, dry the polysaccharide microparticles, place the polysaccharide microparticles dry powder in a concentration of 100 ~ 1000mg / L metal ion solution, shake the adsorption, and adsorb the adsorption After the particles are dried in an oven, they are polysaccharide anticancer materials.
在本发明中, 所说的稀酸为乙酸、 醋酸或盐酸; 金属离子溶液为具有 抗癌活性的多种金属离子的无机酸盐溶液, 如锌离子的硫酸盐、 盐酸盐、 硝酸盐水溶液或为钴离子的硝酸盐等溶液。 In the present invention, the dilute acid is acetic acid, acetic acid or hydrochloric acid; the metal ion solution is an inorganic acid salt solution of various metal ions having anticancer activity, such as zinc ion sulfate, hydrochloride, and nitrate aqueous solutions Or a solution such as nitrate of cobalt ion.
本发明另一方面还涉及一种抗癌组合物,所述组合物包含上述多聚糖 类抗癌材料以及药学上可接受的载体。所述癌症可选自胃癌、肠癌、肝癌、 肺癌、 膀胱癌、 卵巢癌、 胰腺癌、 肾癌、 ***癌。
本文所用的术语 "药学上可接受的"是指当分子本体和组合物适当地 给予动物或人时, 它们不会产生不利的、 过敏的或其它不良反应。 本文所 用的 "药学上可接受的载体"应当与本发明的多聚糖类抗癌材料相容, 即 能与其共混而不会在通常情况下大幅度降低药物组合物的效果。 可作为药 学上可接受的载体或其组分的一些物质的具体例子是糖类, 如乳糖、 葡萄 糖和蔗糖; 淀粉, 如玉米淀粉和土豆淀粉; 纤维素及其衍生物, 如羧甲基 纤维素钠、 乙基纤维素和甲基纤维素; 西黄蓍胶粉末; 麦芽; 明胶; 滑石; 固体润滑剂, 如硬脂酸和硬脂酸镁; 硫酸钙; 植物油, 如花生油、 棉籽油、 芝麻油、 橄榄油、 玉米油和可可油; 多元醇, 如丙二醇、 甘油、 山梨糖醇、 甘露糖醇和聚乙二醇; 海藻酸; 乳化剂, 如 Tween; 润湿剂, 如月桂基硫 酸钠; 着色剂; 调味剂; 压片剂、 稳定剂; 抗氧化剂; 防腐剂; 无热原水; 等渗盐溶液; 和磷酸盐缓冲液等。 Another aspect of the present invention also relates to an anticancer composition, which comprises the above-mentioned polysaccharide anticancer material and a pharmaceutically acceptable carrier. The cancer may be selected from gastric cancer, intestinal cancer, liver cancer, lung cancer, bladder cancer, ovarian cancer, pancreatic cancer, kidney cancer, and prostate cancer. The term "pharmaceutically acceptable" as used herein means that when the molecular body and composition are properly administered to an animal or human, they do not cause adverse, allergic or other adverse reactions. The "pharmaceutically acceptable carrier" used herein should be compatible with the polysaccharide anticancer material of the present invention, that is, it can be blended with it without greatly reducing the effect of the pharmaceutical composition under normal circumstances. Specific examples of some substances that can be used as pharmaceutically acceptable carriers or components thereof are sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and derivatives thereof such as carboxymethyl fiber Sodium cellulose, ethyl cellulose and methyl cellulose; tragacanth powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, Sesame oil, olive oil, corn oil and cocoa butter; polyols such as propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; Coloring agents; Flavoring agents; Tablets, stabilizers; Antioxidants; Preservatives; Pyrogen-free water; Isotonic saline solutions; and phosphate buffers.
本发明的药物组合物可根据需要制成各种剂型, 并可由医师根据患者 种类、 年龄、 体重和大致疾病状况、 给药方式等因素确定对病人有益的剂 量进行施用。 The pharmaceutical composition of the present invention can be formulated into various dosage forms as needed, and can be administered by a physician to determine a dosage that is beneficial to the patient according to factors such as the type of patient, age, weight and general disease status, administration method and the like.
本发明的优点是: The advantages of the invention are:
1)本发明提供了一种在常温、 常压下 (即温和条件下)制备多聚糖类抗 癌材料方法。 制备过程中, 不使用任何有机试剂和表面活性剂, 很好的解 决了多聚糖微粒的分离和纯化问题。 1) The present invention provides a method for preparing a polysaccharide anticancer material at normal temperature and pressure (that is, under mild conditions). In the preparation process, no organic reagents and surfactants are used, which solves the problem of separation and purification of polysaccharide particles.
2)本发明制得的多聚糖类抗癌材料粒径分布均匀, 形态好。 2) The polysaccharide anticancer material prepared by the present invention has a uniform particle size distribution and a good shape.
3)本发明以具有抗癌活性的金属离子与多聚糖为基质制备微粒子,能 够作为抗癌药物广泛应用。 附图说明 3) The invention uses the metal ions and polysaccharides having anticancer activity as a matrix to prepare microparticles, which can be widely used as anticancer drugs. BRIEF DESCRIPTION OF THE DRAWINGS
图 1为实例 1制备所得多聚糖类抗癌材料透视电镜图 (50000倍)。 图 2为实例 2制备所得多聚糖类抗癌材料原子力显微镜图。 Fig. 1 is a perspective electron micrograph (50,000 times) of a polysaccharide anticancer material prepared in Example 1. FIG. 2 is an atomic force microscope image of a polysaccharide anticancer material prepared in Example 2. FIG.
图 3为实例 3制备所得多聚糖类抗癌材料原子力显微镜图。 FIG. 3 is an atomic force microscope image of a polysaccharide anticancer material prepared in Example 3. FIG.
图 4为实例 4制备所得多聚糖类抗癌材料原子力显微镜图。 FIG. 4 is an atomic force microscope image of the polysaccharide anticancer material prepared in Example 4. FIG.
图 5为实例 5制备所得多聚糖类抗癌材料原子力显微镜图。 FIG. 5 is an atomic force microscope image of a polysaccharide anticancer material prepared in Example 5. FIG.
图 6为实例 6制备所得多聚糖类抗癌材料原子力显微镜图。 FIG. 6 is an atomic force microscope image of a polysaccharide anticancer material prepared in Example 6. FIG.
图 7为实例 7多聚糖类抗癌材料处理胃癌细胞 24 h前后透视电镜图
(5000·倍), 其中: a为正常胃癌细胞, b为经多聚糖类抗癌材料作用 24 h后 的胃癌细胞, c为经载金属离子多聚糖类抗癌材料作用 24 h后的胃癌细胞。 具体实施方式 FIG. 7 is a perspective electron micrograph of gastric cancer cells treated with a polysaccharide anticancer material of Example 7 before and after 24 h (5000 · times), where: a is a normal gastric cancer cell, b is a gastric cancer cell treated with a polysaccharide anticancer material for 24 hours, and c is a 24-hour treatment with a metal ion-containing polysaccharide anticancer material. Gastric cancer cells. detailed description
下面结合具体实例进一步说明本发明。在本说明书中,除非另有描述, 所有溶液%均表示为重量 /体积。 The present invention is further described below with reference to specific examples. In this specification, unless otherwise stated, all solutions% are expressed as weight / volume.
实例 1 : Example 1:
20°C磁力搅拌下, 在 0.5%乙酸溶液中加入分子量 llkDa的多聚糖 (购 自浙江省磐安县万昌壳聚糖厂), 配置 0.1%多聚糖酸溶液, 调节溶液 pH值 为 6, 形成微粒子乳液, 3000rpm/min沉淀, 沉淀物蒸馏水漂洗至中性后, 干燥即得多聚糖类抗癌材料。 With magnetic stirring at 20 ° C, a polysaccharide with a molecular weight of llkDa (purchased from Wanchang Chitosan Factory in Pan'an County, Zhejiang Province) was added to a 0.5% acetic acid solution, and a 0.1% polysaccharide acid solution was configured to adjust the pH of the solution to 6 A microemulsion was formed, and the precipitate was precipitated at 3000 rpm / min. After the precipitate was rinsed with distilled water to neutrality, the polysaccharide anticancer material was dried.
上述方法制备的微粒子透视电镜图显示微粒子为较规则球形结构 (见 图 1), 能在 pH值 3〜8内稳定保存。 实例 2: The TEM image of the microparticles prepared by the above method shows that the microparticles have a relatively regular spherical structure (see Figure 1) and can be stably stored at a pH value of 3-8. Example 2:
60°C磁力搅拌下, 在 0.5%醋酸溶液中加入分子量 lOkDa的多聚糖, 配置 5%多聚糖酸溶液,调节溶液 pH值为 7,形成微粒子乳液, 4000rpm/min 沉淀分离, 蒸馏水漂洗至中性, 干燥得多聚糖微粒子, 将多聚糖微粒子干 粉置于 600mg/L钴离子溶液中, 300rpm/min振荡吸附 (30 min-24 h), 吸附 完全后, 将吸附后多聚糖微粒子置于烘箱 60°C烘干, 即得多聚糖类抗癌材 料。 Under a magnetic stirring at 60 ° C, a polysaccharide with a molecular weight of lOkDa was added to a 0.5% acetic acid solution, and a 5% polysaccharide acid solution was configured. The pH of the solution was adjusted to 7 to form a microemulsion. The precipitate was separated at 4000 rpm / min, and rinsed with distilled water to Neutral, dried polysaccharide microparticles, put the polysaccharide microparticles dry powder in 600mg / L cobalt ion solution, shake adsorption at 300rpm / min (30 min-24 h), after the adsorption is completed, the polysaccharide microparticles after adsorption Put it in an oven at 60 ° C to dry, which is polysaccharide anti-cancer material.
上述方法制备的载钴微粒子中钴含量约为 10%,原子力显微镜图显示 微粒子为较规则球形结构 (见图 2), 能在 pH值 3〜8内稳定保存。 实例 3: The cobalt content in the cobalt-loaded microparticles prepared by the above method is about 10%, and the atomic force microscope image shows that the microparticles have a relatively regular spherical structure (see Figure 2) and can be stably stored at a pH value of 3-8. Example 3:
30°C磁力搅拌下, 在 1%盐酸溶液中加入分子量 20kDa的多聚糖, 配 置 2%多聚糖酸溶液, 调节溶液 pH值为 8, 形成微粒子乳液, 6000rpm/min 沉淀, 沉淀物蒸馏水漂洗至中性后, 干燥即得多聚糖类抗癌材料。 Under 30 ° C magnetic stirring, add a polysaccharide with a molecular weight of 20 kDa to a 1% hydrochloric acid solution, configure a 2% polysaccharide acid solution, adjust the pH of the solution to 8 to form a microemulsion, precipitate at 6000 rpm / min, rinse the precipitate with distilled water After neutralization, the polysaccharide anticancer material is dried.
上述方法制备的微粒子原子力显微镜图显示微粒子为球形结构 (见图 3), 能在 pH值 3〜8内稳定保存。
实例 4: The atomic force microscope image of the microparticles prepared by the above method shows that the microparticles have a spherical structure (see Figure 3), and can be stably stored at a pH value of 3 to 8. Example 4:
35Ό磁力搅拌下, 在 1%乙酸溶液中加入分子量 30kDa的多聚糖, 配 置 1.5%多聚糖酸溶液,调节溶液 pH值为 9,形成微粒子乳液, 3000rpm/min 沉淀, 沉淀物蒸馏水漂洗至中性后, 干燥即得多聚糖类抗癌材料。 Under 35 ° magnetic stirring, polysaccharide with a molecular weight of 30 kDa was added to a 1% acetic acid solution, and a 1.5% polysaccharide acid solution was configured. The pH of the solution was adjusted to 9 to form a microemulsion. The precipitate was precipitated at 3000 rpm, and the precipitate was rinsed with distilled water. After sex, the polysaccharide anticancer material is dried.
上述方法制备的多聚糖类抗癌材料原子力显微镜图显示微粒子为较 规则球形结构 (见图 4), 能在 pH值 3〜8内稳定保存。 实例 5 : Atomic force microscopy of the polysaccharide-based anticancer material prepared by the above method shows that the microparticles have a relatively regular spherical structure (see Figure 4), and can be stably stored at a pH value of 3-8. Example 5:
25Ό磁力搅拌下, 在 0.5%乙酸溶液中加入分子量 50kDa的多聚糖, 配置 1%多聚糖酸溶液,调节溶液 pH值为 6,形成微粒子乳液, 4000rpm/min 沉淀分离, 蒸馏水漂洗至中性, 干燥得多聚糖微粒子, 将多聚糖微粒子干 粉置于 600mg/L钴离子溶液中, 300rpm/min振荡吸附 (30 min-24 h), 吸附 完全后, 将吸附后多聚糖微粒子置于烘箱 60°C烘干, 即得多聚糖类抗癌材 料。 Under a magnetic stirring of 25 ,, add a polysaccharide with a molecular weight of 50 kDa to a 0.5% acetic acid solution, configure a 1% polysaccharide acid solution, adjust the pH value of the solution to 6 to form a microemulsion, separate by precipitation at 4000 rpm / min, rinse with distilled water to neutrality To dry the polysaccharide microparticles, place the polysaccharide microparticles dry powder in a 600mg / L cobalt ion solution, and shake and adsorb (30 min-24 h) at 300rpm / min. After the adsorption is complete, place the polysaccharide microparticles after the adsorption. Drying in an oven at 60 ° C is a polysaccharide anticancer material.
上述方法制备的多聚糖类抗癌材料其中钴含量约为 20%, 原子力显微 镜显示多聚糖类抗癌材料为较规则球形结构 (见图 5), 能在 pH值 3〜8内 稳定保存。 实例 6: The polysaccharide anticancer material prepared by the above method has a cobalt content of about 20%. Atomic force microscopy shows that the polysaccharide anticancer material has a relatively regular spherical structure (see Figure 5), and can be stably stored at a pH value of 3 to 8. . Example 6:
30°C磁力搅拌下, 在 0.5%醋酸溶液中加入分子量 60kDa的多聚糖, 配置 2%多聚糖酸溶液,调节溶液 pH值为 7,形成微粒子乳液, 6000rpm/min 沉淀分离, 蒸馏水漂洗至中性, 干燥得多聚糖微粒子, 将多聚糖微粒子干 粉置于 800mg/L锌离子溶液中, 400rpm/min振荡吸附 (30 min-24 h), 吸附 完全后, 将吸附后多聚糖微粒子置于烘箱 40°C烘干, 即得多聚糖类抗癌材 料。 Under 30 ° C magnetic stirring, add a polysaccharide with a molecular weight of 60 kDa to a 0.5% acetic acid solution, configure a 2% polysaccharide acid solution, adjust the pH of the solution to 7 to form a microemulsion, precipitate and separate at 6000 rpm / min, rinse with distilled water to Neutral, dried polysaccharide microparticles, put the polysaccharide microparticles dry powder in 800mg / L zinc ion solution, shake adsorption at 400rpm / min (30 min-24 h), after the adsorption is complete, the polysaccharide particles after adsorption will be absorbed Dry in an oven at 40 ° C, which is polysaccharide anti-cancer material.
上述方法制备的多聚糖类抗癌材料其中锌离子含量约为 8%, 多聚糖 类抗癌材料原子力显微镜图显示形态较好球形结构 (见图 6),能在 pH值 3〜 8内稳定保存。 实例 7: The polysaccharide-based anticancer material prepared by the above method has a zinc ion content of about 8%, and the atomic force microscope image of the polysaccharide-based anticancer material shows a good spherical structure (see Figure 6), which can be within pH 3 to 8. Stable. Example 7:
胃癌细胞株 MGC803置于 37°C, 5%( 02细胞培养箱中用 RPMI-1640
培养液进行培养, 每 2 d更换细胞培养液, 待细胞长满瓶底后再传代。 细胞悬液培养 24 h后, 弃原培养液, 加入新鲜培养液 100 L, 测定 孔加 11 μL不同浓度多聚糖类抗癌材料与阳性对照药物 (多聚糖), 对照孔 加入等量 RPMI-1640培养液,各设 4个重复。重新放入培养箱培养 24、 48、 72、 120h后取出,每孔加入 ΜΤΤ20 μί,继续培养 4 h,每孔加入 DMSO100 震荡 10 min, 酶标仪 490 nm为检测波长, 测各孔 OD值, 计算抑制 率。 抑制率 >30%为药敏阳性, 反之阴性。表 1给出了多聚糖类抗癌材料对 胃癌细胞毒性 MTT试验。 Gastric cancer cell line MGC803 was placed at 37 ° C in a 5% (0 2 cell incubator with RPMI-1640 The culture medium was cultured, and the cell culture medium was changed every 2 days, and the cells were passaged after the bottom of the bottle was full. After culturing the cell suspension for 24 hours, discard the original medium, add 100 L of fresh medium, add 11 μL of polysaccharide anticancer materials with different concentrations and the positive control drug (polysaccharide) to the assay wells, and add equal amounts of RPMI to the control wells. -1640 culture medium, each set of 4 replicates. Put it back into the incubator for 24, 48, 72, and 120 hours and take it out. Add MTTT 20 μL to each well, continue to incubate for 4 h, add DMSO100 to each well and shake for 10 min. Use a microplate reader at 490 nm as the detection wavelength. Calculate the inhibition rate. Inhibition rate> 30% is positive for drug sensitivity, otherwise it is negative. Table 1 shows the toxicity MTT test of polysaccharide anticancer materials on gastric cancer cells.
抑制率 =(1-药物 OD值 /对照 OD值) X 100% Inhibition rate = (1-OD value of drug / OD value of control) X 100%
表 1 多聚糖微粒抗癌材料对胃癌细胞毒性 MTT试验 Table 1 Toxicity of polysaccharide microparticle anticancer materials to gastric cancer cells MTT test
"与(^g/ml组 1%乙酸相比 P<0.01, "P <0.01 compared with 1% acetic acid in the (^ g / ml group,
++ 与多聚糖 (100)组相比 P<0.01, + + Compared with the polysaccharide (100) group, P <0.01,
"多聚糖" 为多聚糖醋酸溶液, 其余为不同浓度的多聚糖微粒子水溶 液。 "Polysaccharide" is a polysaccharide acetic acid solution, and the rest are polysaccharide microparticle aqueous solutions of different concentrations.
根据表 1所示结果可以看出, 与未经本发明方法处理的多聚糖醋酸溶 液相比, 本发明获得的多聚糖微粒子的抑制率显著提高。 尤其在多聚糖浓 度相同 (100微克 /毫升)的情况下, 多聚糖微粒子对癌细胞的抑制率比未经
处理的多聚糖阳性对照提高 34.3 %。 According to the results shown in Table 1, it can be seen that compared with the polysaccharide acetic acid solution not treated by the method of the present invention, the inhibition rate of the polysaccharide fine particles obtained by the present invention is significantly improved. Especially at the same polysaccharide concentration (100 μg / ml), the inhibitory rate of polysaccharide microparticles on cancer cells was higher than that without The treated glycan positive control increased by 34.3%.
另外,使多聚糖类抗癌材料作用胃癌细胞 24h后,用 10%戊二醛固定, 0.1M磷酸缓冲液漂洗 3次, 1%锇酸后 2h,丙酮加 Epon 812逐级渗透, Epon 812环氧树脂原位包埋。 LKB超薄切片机切片, 醋酸双氧铀和椽酸铅双染 色, JEM-1200EX型透视电镜观察。 结果如图 7所示。 In addition, after applying polysaccharide anticancer materials to gastric cancer cells for 24 hours, they were fixed with 10% glutaraldehyde, rinsed with 0.1M phosphate buffer 3 times, and 2 hours after 1% gallic acid, acetone plus Epon 812 was gradually penetrated, and Epon 812 Epoxy resin is embedded in situ. LKB ultra-thin microtome section, double staining of uranyl acetate and lead osmium, JEM-1200EX type TEM observation. The results are shown in Figure 7.
根据图 7可以看出, 对照组细胞 (图 7a)胞体光滑, 细胞膜表面布有微 絨毛。 细胞器正常, 线粒体呈圆形或卵形分布, 嵴膜清楚, 核膜完整, 染 色质均匀分散。 经多聚糖微粒处理后 (图 7b和 7c), 细胞空泡变, 细胞膜破 裂, 核被膜消失, 多数细胞器破碎, 线粒体肿胀, 核浓缩, 核仁由网状转 呈环状结构。 尽管本发明描述了具体的例子,但是有一点对于本领域技术人员来说 是明显的, 即在不脱离本发明的精神和范围的前提下可对本发明作各种变 化和改动。 因此, 所附权利要求覆盖了所有这些在本发明范围内的变动。
According to Fig. 7, it can be seen that the cells of the control group (Fig. 7a) have smooth cell bodies and the surface of the cell membrane is covered with microvilli. The organelles are normal, the mitochondria are round or oval, the diaphragm is clear, the nuclear membrane is intact, and the dye quality is evenly dispersed. After treatment with polysaccharide microparticles (Figures 7b and 7c), the vacuoles of the cells changed, the cell membrane broke, the nuclear envelope disappeared, most of the organelles were broken, the mitochondria were swollen, and the nuclei were condensed. Although the present invention describes specific examples, it is obvious to those skilled in the art that various changes and modifications can be made to the present invention without departing from the spirit and scope of the present invention. It is therefore intended to cover in the appended claims all such changes that are within the scope of this invention.