【作者】 李伟；

【导师】 屠幼英；

【作者基本信息】 浙江大学 ， 茶学， 2006， 硕士

【摘要】 本论文用茶黄素复合物及单体对多种癌细胞进行抑制作用研究,并且首次将抗坏血酸与茶黄素进行人肺癌细胞A549的协同抑制作用试验。此外,分析了多种植物提取物对人肺癌细胞的抑制作用效果,主要结果概括如下:用MTT法检测了浓度分别为1、0.25、0.063、0.016、0.039mg/ml的TFs、TFDG、TF2B和UC对肝癌BEL-7402细胞和胃癌MKN-28细胞的生长抑制效应。结果显示这些化合物对上述细胞有明显的生长抑制效应,其抑制率随药物浓度的升高和作用时间延长而增加。TFs、TFDG、TF2B和UC与BEL-7402细胞共同培养48h后测得IC₅₀分别为0.18mM、0.16mM、0.11mM和0.08mM和胃癌MKN-28细胞的IC₅₀分别为0.58mM、0.25mM、0.14mM和0.22mM。 通过EGCG、GSE、PBE、TFDG、TF和TFs的抗人肺癌A549细胞体外实验,结果表明除槲寄生的作用较小以外其他化合物均有很强促进人肺癌细胞凋亡的作用,并且EGCG的效果为最强,顺序为EGCG、TFs>GSE>PBE、TFDG>TF。 细胞凋亡实验表明,单独的抗坏血酸对A549细胞的抑制几乎没有作用,但它可以显著的提高茶黄素类物质对该细胞的抑制效果。其中协同效果与单体TFc最大,抑制率高达84.28%。茶黄素各单体与浓度为2.50～6.25μM抗坏血酸协同抑制肺癌细胞A549时最少可以提高20%的抑制率。其中最好的协同抑制效果存在于TFc和抗坏血酸之间,。茶黄素单体与抗坏血酸的协同普遍高于EGCG与AA的协同。但不同浓度的抗坏血酸与单体TF协同效果差别不明显。 电镜观察TFc和TFc与AA协同诱导的A549细胞的凋亡现象,观察到细胞核发生固缩,染色质浓缩边集和产生由膜状物包裹内溶物的凋亡小体。并且协同诱导时凋亡效果最明显。 DNA凝胶电泳结果显示,在此处理中可非常明显的看到细胞凋亡。凋亡细胞的DNA断裂点均有规律的发生在核小体之间,出现了180—200bp整倍体的DNA片断,而坏死细胞的DNA断裂点为无规律的杂乱片断。还可以清晰的看到标志细胞凋亡的梯形条带（DNA ladders）。 综上所述,茶黄素及其单体可以诱导多种肿瘤细胞凋亡并且与抗坏血酸有良好的协同作用。由于细胞膜有不同程度的损伤,细胞膜是茶黄素、抗坏血酸或两者相互作用时对细胞损伤的主要作用位点。更多还原

【Abstract】 In the present study inhibitions of theaflavins and its monomers againstthe growth of four human cancercells in vitro were concerned. Synergistic inhibition of theaflavins and ascorbic acid against the growth of A549 cells was studied. Beside these, we determined some effects of some natural products on human cancer cells A549. The results as below: The inhibition effects of TFs, TFDG, TF2B and UC on the growth of human liver cancer BEL-7402 cells, gastric cancer MKN-28 cells were investigated by MTT assay. The results showed that these four compounds significantly inhibited the growth of both two kinds of cancer cells, and effective in inhibition rate in a dose dependent manner. The IC₅₀ values were 0.18, 0.16, 0.11 and 0.08mM on BEL-7402 for TFs, TFDG, TF2B and UC, and 0.58, 0.25, 0.14 and 0.22 mM on BEL-7402 for TFs, TFDG, TF2B and UC, respectively.EGCG, GSE, PBE, TFDG and TFs also showed significant inhibitions on the growth of A549 except VCE. The rank of inhibition ability of each compoundagainst A549 in vitro as following: EGCG, TFs >GSE>PBE, TFDG >TF.In this study We got the different result with previous research, ascorbic acid has no effect on the growth of A549 cells. It was found that ascorbic acid could increased the inhibition of theaflavins at least 20% higher to compare with controls against the growth of A549 cells. TFc showed thehighest synergistic effect with ascorbic acid in all treatments, .however, ascorbic acid no good positive action withEGCG. Whereas, there is no significant different inhibition on the growth of A549 cells when same compound to combined with different concentration of ascorbic acid.The changes of apoptosis induced by TFc and TFc with ascorbic acid wereobserved with electron microscopy. We discerned the typical apoptotic morphology including and condensation into dense granules, and the formation of apoptotic bodied including condensation into dense granules and blocks.A typical DNA degradation associated with apoptosis was determined by DNA agarose electrophoresis. Typical DNA ladders was very clear, this is differ from necrotic.In conclusion, theaflavins and theaflavin monomers can induce the apoptosis of cancer cells. Ascorbic acid has the synergistic effect with theaflavinsagainst the growth of A549 cells. We found characters of apoptosis and damages of cytoplasm memberance in A549 cells culture system, and cytoplasm memberance might be the important target point of theaflavins, ascorbic and their complex.更多还原