壳聚糖及其衍生物对肝癌细胞SMMC7721生长的抑制作用

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。

Antigrowth effects of chitosan and its derivatives on human hepatocellular carcinoma cell line SMMC7721

BACKGROUND: Different derivatives of chitosan with different molecular weights or degrees of deacetylation show different anti-tumor effects.OBJECTIVE: To study the inhibition effect of water-soluble chitosan and its derivatives, such as sulfonated chitosan, carboxymethyl chitosan and chitooligosaccharides for the growth of hepatocellular carcinoma cell line SMMC7721.DESIGN, TIME AND SETTING: Controlled experiments based on observation were carried out in Jiangxi Institute of Digestive Disease (Nanchang, Jiangxi, China) from January 2004 to December 2006.MATERIALS: Hepatoma cell line SMMC7721 was provided by Jiangxi Institute of Digestive Disease (China). 85.5% deacetylated chitooligosaccharides and 85% deacetylated water-soluble chitosan were produced by Jinan Haidebei Ocean Biological Engineering Co., Ltd (China); Carboxymethyl chitosan and 88.5% deacetylated chitosan were the products of Shanghai Qisheng Biological Products Co., Ltd (China).METHODS: Sulfonated chitosan was prepared using 88.5% deacetylated chitosan and chlorosulfonic acid-formamide, and then was detected with infrared spectroscopy in the Detection Analysis and Test Center, East China University of Science and Technology. SMMC7721 cells in the log phase were inoculated into 96-well culture plates, which were then added with water-soluble chitosan, sulfonated chitosan, carboxymethyl chitosan and chitooligosaccharides with the final concentrations of 25, 50, 100, 200, 400 and 800mg/L. This test was repeated for 3 times, while the control group was also set each time. After 72 hours of routine culture, MTT solution was added into each well and inoculated for another 4 hours. After the culture was terminated, dimethyl sulfoxid was added. The absorbance value of each well was measured at 490nm wavelength on a microplate reader. Three tests were measured to obtain the mean value. Also the inhibition rate was calculated.MAIN OUTCOME MEASURES: Growth inhibition effect of chitosan and its derivatives on the hepatoma cell line SMMC7721.RESULTS: Among the chitosan and its derivates at four kinds of concentrations, water-soluble chitosan and sulfonated chitosan could significantly inhibit the growth of SMMC7721 cells (P<0.001), and the effect was the most significant in the case of sulfonated chitosan. Treatment with water-soluble chitosan and sulfonated chitosan at the concentration of 50mg/L could inhibit the growth of SMMC7721 cells in a dose-dependent manner, and reached a peak at the concentration of 400mg/L and 800mg/L, respectively. Carboxymethyl chitosan and chitooligosaccharides showed no growth inhibition effect (P>0.05).CONCLUSION: Water-soluble chitosan and sulfonated chitosan have significant antigrowth effects on hepatoma carcinoma cells, while carboxymethyl chitosan and chitooligosaccharides are ineffective.