【作者】 迟万好；

【导师】 朱睦元； 曹明富；

【作者基本信息】 浙江大学 ， 遗传学， 2002， 硕士

【摘要】 茶多酚（tea polyphonels，TPs）是以茶叶为原料提取出来的一类多酚类化合物，除含有酚酸、黄酮、黄酮醇和花色素外，其主要成份是茶儿茶素（黄烷醇类），约占TPs的60％-80％。茶儿茶素主要包括表儿茶素＜（-）-epicatechin，EC＞、表儿茶素没食子酸酯＜（-）-epicatechin-3-gallate，ECG＞、没食子酸儿茶素＜（-）-epigallocatechin，EGC＞、表没食子儿茶素没食子酸酯＜（-）-epigallocatechin-3-gallate，EGCG＞等单体。其中以EGCG的含量最高，占儿茶素的50％左右。对茶及TPs的药效研究已有大量文献报道。本研究为探明TPs对细胞的影响及引发细胞凋亡的机理，采用细胞生物学、生化与分子生物学等技术测定了TPs及其单体EGCG对体外培养人不同肿瘤细胞株的细胞毒作用；EGCG诱导D6细胞凋亡的作用，并且根据凋亡检测的不同指标来探讨EGCG诱导D6细胞凋亡的作用机理。 细胞存活率实验的结果表明：TPs和EGCG可以抑制四种肿瘤细胞生长，并在一定剂量范围（TPs浓度为0～500μg/mL，EGCG浓度为0～625μg/mL）内呈浓度和时间依赖性；不同组织来源的肿瘤细胞对TPs和EGCG的敏感性不同：D6、SGC-7901、SMMC-7721、PC-3细胞的TPs半抑制浓度(IC₅₀)分别为71.1μg/mL、100.5μg/mL、93.8μg/mL、116.6μg/mL，EGCG的IC₅₀分别为58.6μg/mL、114.3μg/mL、114.3μg/mL、90.8μg/mL；TPs和EGCG对同一组织来源的正常细胞和肿瘤细胞有不同的作用：TPs对人肺癌细胞（D6）和人胚肺细胞（WI-38）的IC₅₀分别为71.1μg/mL和1786.7μg/mL，EGCG对D6和WI-38的IC₅₀分别为58.6μg/mL和2177.4μg/mL；低浓度的TPs和EGCG促进正常细胞（WI-38）的生长，而高浓度却有抑制生长的作用，并呈浓度依赖性。高浓度的TPs和EGCG对肿瘤细胞的抑制作用不如低浓度好。 EGCG作用D6细胞后采用Hoechst33258荧光染料染色并且在荧光显微镜下观察，发现随EGCG作用浓度的增大，细胞出现染色体边集、DNA断裂、 染色质环化等现象，而对照细胞的细胞核质呈现均一的颜色。处理后的D6细 胞，DNA琼脂糖凝胶电泳出现典型的DNA梯状条带。以上结果表明，EGCG 在一定浓度和时间条件下，可以诱导 D6细胞凋亡，并且以在浓度为 80 V g／InL 作用48h效果最好。通过检测EGCG处理D6细胞后ROS的变化，发现80 H g／InL EGCG诱导 D6细胞凋亡时产生的 ROS是对照的 188．9％，有极显著差异， 说明ROS参与了EGCG诱导D6细胞的凋亡，D6细胞内ROS水平升高与GST 无关。实验发现，在 D6凋亡的信号传导过程中，没有 C卿ase 8和 BCL1蛋白 的参与。EGCG诱导D6细胞凋亡可能是EGCG引发D6细胞内ROS水平的升 高，进而经由线粒体途径引发细胞凋亡。更多还原

【Abstract】 Tea (Camellia sinensis) is one of the most common beverages consumed worldwide. TPs (tea pplyphonels) are most abundant in green tea and they are mostly in the form of flavanols commonly known as catechins which include (-)-epicatechm(EC),(-)-epicatecbJn-3-gallate(ECG),(-)-epigallocatechin(ECG),(-)-epigallocatechin-3-gallate (EGCG). TPs demonstrated profound biochemical and pharmacological including antioxidant activities, modulation of carcinogen matabolism and inhibition of cell proliferation. Recently, many experiments proved that TPs could induce apoptosis in various cancer cells, hi the precent study, the action of TPs and EGCG inhibited various human caner cells in vitro were investigated using human lung cancer D6 cells, human stomach cancer SGC-7901cells, human liver cancer cells SMMC-7721 cells and human prostate cancer PC-3 cells as a model.In MTT assay, IPs and EGCG can inhibit the growth of four kinds of cancer cells in lower concentration (TPs and EGCG is 0-500 U g/mL and 0-625 u g/mL, respectively) in concentration- and time-dependent. Different effects of inhibition on cells were depent on the kind of cell. ICso values of TPs and EGCG against D6, SGC-7901, SMMC-7721, PC-3 are 71.1 u g/mL, 100.5 u g/mL, 93.8 u g/mL, 116.6 ug/mL and 58.6 u g/mL, 114.3 u g/mL, 114.3 u g/mL, 90.8 u g/mL respectively. TPs and EGCG have different effects on normal and cancer cells derived of the same orgnize. ICso values of TPs and EGCG against D6 and WI-38 are 71.1 u g/mL, 1786.7 u g/mL and 58.6 U g/mL, 2177.4 u g/mL respectively. Lower concentrition of TPs and EGCG increased the number of WI-38 and higher concentrition of TPs and EGCG also can inhibit the growth of WI-38 cell and is concentration-dependent. The inhibition of lower concentrition of TPs and EGCG is stronger than the inhibition of higher concentrion for cancer cells.Morphological changes in D6 cells after treated with EGCG showed in fluoresent microscopy dyed with Hoechst33258 for lOmin. Typical apoptotic characteristics of D6 cells were determined by fluorescent microscopy and DNA fragment electrophoresis after the cells were treated with EGCG for 80 u g/mL for 48h. The ROS level in cells treated with 80 u g/mL EGCG increases 88.9% than that in control cells indicating that ROS is connective with apoptosis in D6 cells. The results of Caspase 8 test and GST test indicated that they don’t join in apoptosis in D6 cells. Further experiments examine the expression of BCL-2 by Western blot and no bands were observed. The mechanism of apoptosis induced by EGCG in D6 cells may be as the following: EGCG induce the increase of ROS level in D6 cells, and the latter induce apoptosis in D6 cells by mitochondria signal transduction pathway.更多还原