【作者】 张亮；

【导师】 单吉贤；

【作者基本信息】 中国医科大学 ， 肿瘤学， 2005， 硕士

【摘要】 目的 姜黄素（curcumin,Cur）是来源于姜科植物姜黄中的一种酚性色素。目前发现其具有抗氧化、抗炎、抗凝、降血脂、抗动脉粥样硬化及抗肿瘤的功效。关于抗肿瘤作用的研究已成为当今研究的热点,因姜黄素抗肿瘤谱广、毒副作用小且价格低廉,所以是一种具有广泛应用前景的抗肿瘤新药。关于姜黄素抗肿瘤治疗的研究还主要集中在肿瘤化学预防方面,美国国立肿瘤所已将其列为第3代癌化学预防药。国内外现有研究发现姜黄素具有: 1、抗亚硝化作用;2、抗NO作用;3、抗鸟氨酸脱羧酶活性及DNA合成;4、抗DNA的氧化损伤;5、抗血管生成;6、诱导肿瘤细胞分化,但其确切的抗肿瘤作用机制仍不十分明确。本实验是想通过姜黄素在体外对大肠癌CX-1细胞株的抗肿瘤作用研究,探讨姜黄素抗大肠癌的作用机理及其分子机制。 实验方法 人大肠癌细胞株CX-1维护于含10%胎牛血清、谷氨酰胺和青、链霉素的RPMI1640培养液中,置于37℃、含5%CO₂及饱和湿度的培养箱中培养,待细胞处于对数生长期时进行实验;采用细胞生长抑制试验（MTT法）检测不同浓度姜黄素对大肠癌CX-1细胞的生长抑制作用,设立实验组和对照组。通过酶标仪于570nm波长测吸光度（A）值,肿瘤细胞的抑制率（%）=（1-实验组A值/阴性对照组A值）×100%;一定浓度姜黄素处理到预定时间后收集培养基中的细胞,制成1×10⁷/1细胞悬液,经丫啶橙染色后,在荧光显微镜下观察细胞核的形态变化;光镜下观察对照组和实验组CX-1细胞的生长情况、形态学改变及凋亡过程;透射电镜技术观察对照组和实验组CX-1细胞的超微结构,特别是观察实验组细胞的凋亡结构;通过流式细胞仪测定一定浓度姜黄素处理CX-1细胞不同时间的凋亡率,并更多还原

【Abstract】 ObjectiveCurcumin is the major effective component of curcuma, which is a kind of tradition Chinese medicine. It has been paid more attention to curcumin recently for its specific function of anti - oxidation , anti - inflammation ,lipid - regulation ,anti - thrombin,anti - arteriosclerosis and anti - tumor. The study about its anti - tumor function has already become the highlight of today. For its broad -spectrum of anti - tumor ,little side effect and low price, curcumin is a new kind of anti - tumor drug with bright future. The study concerning curcumin anti -tumor effect is mainly focused on tumor chemical prevention, and America national tumor institute has ranked it as the third generation drug for cancer chemical prevention. Present studies suggest curcumin has the following functions: 1. anti - nitrolysation; 2. anti - NO; 3. anti - synthesization of DNA 4. anti - oxidation damage of DNA; 5. anti - vessel generation; 6. inducing tumor cell differentiation ; but the exact mechanism of anti — tumor is not clear. This study is designed to investigate the possible anti - tumor mechanism and molecular mechanism of curcumin through the trial in vitro in human colon cancer CX -1 cells.MethodsHuman colon cancer cell group CX -1 was sustained in the RPMI1640 culture medium containing 10% serum of fetal calf , glutamine , penicillin and streptomycin, and cultured in the box with 5% CO₂ and saturated moisture. When cells developed into logarithmic growth stage, the experiment was performed. The inhibition of cells growth induced by various concentration of curcumin indifferent course was analyzed by using MIT assay, at the same time experiment group and control group were both set, then using machine to detect light - absorption rate at wave 570nm. Inhibitory rate of tumor cell （ % ） = （1 - A value of experiment group /A value of negative control group） 100% ; Certain concentration curcumin was processed till the schedule time when the cells in the culture medium were collected, and made into cell — suspending fluid with concentration of 1 107/L. The nuclei were stained by acridine orange fluorescent staining method and the morphological changes of the cell nuclei were examined under fluorescence microscope; development,morphological changes and apop-totic process of CX -1 cells for both control and experiment groups were detected under light microscope; Super - microstructure of CX - 1 cells were observed under transmission electron microscope, especially for the apoptotic structure of the cells in experimental group; The cell apoptosis ratio of CX - 1 processed with curcumin in different times were measured by flow cytometry a-nalysis and compared with control group. It has already revealed that there is an relationship between the processing time of curcumin and the apoptosis rate; The quantity of apoptosis - inhibiting protein Bel - 2 and apoptosis - promoting protein bax were evaluated by immuno - histochemistiy method both before and after processed with certain concentration curcumin; positive and negative contrast were set in the trial.ResultsGrowth inhibitory rates of CX - 1 cells by curcumin were significantly different in dose - dependent manners. Human colon cancer CX - 1 cells were processed respectively with 1,2,4,8,16,32ug/ml curcumin and 72 hours later the inhibitory rates revealed to be 20.67% ,40.97% ,67.75% ,83.05% ,85.28% , 91.24% , then compared with controlled group P <0.01; Chromatin condensation and nuclear fragmentation were seen under fluorescence microscope in cells treated with acridine orange fluorescent staining method. The apoptotic cells were observed under transmission electron microscope and elected by flow cytometry. Another apex of sub - diploid appeared in front of G0/G1 apex, and更多还原