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背景:壳聚糖的不同衍生物、不同的分子质量和脱乙酰度对抗肿瘤的作用有所不同。目的:观察水溶性壳聚糖及其3种衍生物磺化壳聚糖、羧甲基壳聚糖、壳寡糖对肝癌细胞株SMMC7721生长的抑制作用。设计、时间及地点:对比观察实验,于2004-01/2006-12在江西省消化疾病研究所完成。材料:肝癌细胞株SMMC7721由江西省消化疾病研究所传代培养。85.5%脱乙酰度壳寡糖和85%脱乙酰度水溶性壳聚糖为济南海得贝海洋生物工程有限公司产品;88.5%脱乙酰度壳聚糖和羧甲基壳聚糖为上海其胜生物制品有限公司产品。方法:①磺化壳聚糖的制备和鉴定:88.5%脱乙酰度壳聚糖与氯磺酸-甲酰胺磺化试剂制得磺化壳聚糖。由华东理工大学分析测试中心采用红外光谱分析检测。②MTT试验:将对数生长期的SMMC7721肝癌细胞株接种于96孔细胞培养板中,分别加入6个质量浓度(25,50,100,200,400,800mg/L)的水溶性壳聚糖、磺化壳聚糖、羧甲基壳聚糖和壳寡糖,并设空白对照组。常规培养72h后,加MTT溶液,孵育4h后终止培养,加二甲基亚砜,酶标仪测490nm波长A值。重复3次试验,取均值,计算抑制率。主要观察指标:不同质量浓度壳聚糖及其衍生物对肝癌细胞的生长抑制率。结果:在4种不同壳聚糖及其衍生物中,水溶性壳聚糖和磺化壳聚糖可显著抑制肝癌细胞的生长(P<0.001),其中以磺化壳聚糖最为显著,二者在质量浓度为50mg/L时就可抑制肝癌细胞的生长,并呈剂量依赖关系,在400mg/L和800mg/L时达到高峰。羧甲基壳聚糖和壳寡糖对肝癌细胞无生长抑制作用(P>0.05)。结论:水溶性壳聚糖和磺化壳聚糖对肝癌细胞具有显著的生长抑制作用,羧甲基壳聚糖和壳寡糖对肝癌细胞无生长抑制作用。
Background: Different derivatives of chitosan, different molecular weights and degrees of deacetylation have different antitumor effects. Objective: To observe the inhibitory effect of water-soluble chitosan and its three derivatives sulfonated chitosan, carboxymethyl chitosan and chitooligosaccharides on the growth of hepatoma cell line SMMC7721. DESIGN, TIME AND SETTING: The comparative observation experiment was performed at the Institute of Digestive Disease of Jiangxi Province from January 2004 to December 2006. Materials: Liver cancer cell line SMMC7721 was subcultured by Institute of Digestive Diseases of Jiangxi Province. 85.5% deacetylated chitooligosaccharides and 85% deacetylated water-soluble chitosan are products of Jinan Highland Marine Engineering Co., Ltd. 88.5% deacetylated chitosan and carboxymethyl chitosan are the best ones in Shanghai Biological Products Co., Ltd. products. Methods: ① Preparation and identification of sulfonated chitosan: 88.5% deacetylated chitosan and chlorosulfonic acid - formamide sulfonated reagent obtained sulfonated chitosan. East China University of Technology Analysis and Testing Center using infrared spectroscopy detection. ② MTT test: SMMC7721 hepatoma cell line in logarithmic growth phase was inoculated into 96-well cell culture plate and added with 6 kinds of water-soluble chitosan, sulfonated chitosan, carboxylated sulfonated chitosan and carboxymethylcellulose at mass concentration (25,50,100,200,400,800mg / L) Methyl chitosan and chitooligosaccharides, and set a blank control group. Conventional culture 72h, add MTT solution, incubated for 4h after termination of culture, plus dimethyl sulfoxide, microplate reader measured 490nm wavelength A value. Repeat the test 3 times, take the average, calculate the inhibition rate. MAIN OUTCOME MEASURES: Growth inhibitory rate of hepatocarcinoma cells with different concentrations of chitosan and its derivatives. Results: Among the four chitosan and its derivatives, water-soluble chitosan and sulfonated chitosan significantly inhibited the growth of hepatocarcinoma cells (P <0.001), of which sulfonated chitosan was the most prominent, In the concentration of 50mg / L can inhibit the growth of liver cancer cells in a dose-dependent manner, peaked at 400mg / L and 800mg / L. Carboxymethyl chitosan and chitooligosaccharides had no growth inhibitory effect on hepatoma cells (P> 0.05). CONCLUSION: Water-soluble chitosan and sulfonated chitosan have significant inhibitory effect on hepatocarcinoma cells. Carboxymethyl chitosan and chitooligosaccharides have no inhibitory effect on hepatocarcinoma cells.