EGCG诱导口腔鳞癌Tca8113细胞凋亡及其机制研究

发布时间：2018-06-07 06:58

  本文选题：表没食子儿茶素没食子酸酯 + caspase-8　； 参考：《皖南医学院》2014年硕士论文

【摘要】：目的：探讨绿茶提取物表没食子儿茶素没食子酸酯（epigallocathechin-3-gallate,EGCG）诱导口腔鳞癌Tca8113细胞凋亡及其可能机制研究。以期为EGCG在口腔鳞癌预防和治疗中提出理论依据。 方法：不同剂量的EGCG（0、25、50、75、100、125mg/L）分别作用Tca8113细胞24h、48h、72h,MTT法观察该药物对Tca8113细胞生长的影响，并计算细胞增殖抑制率。倒置显微镜下观察EGCG（0、50、75、100mg/L）处理48h后Tca8113细胞的形态学变化。AO/EB荧光染色法检测不同剂量的EGCG（0、25、50、75、100mg/L）分别作用Tca8113细胞48h后荧光显微镜下细胞的形态学变化。Hochest33258染色法检测不同剂量EGCG（0、25、50、75、100、125mg/L）对Tca8113细胞凋亡的影响。流式细胞仪检测EGCG（0、50、75mg/L）干预48h后Tca8113细胞周期的改变。透射电镜下观察加药组与阴性对照组之间的区别，其中加药组是否出现凋亡现象。免疫细胞化学SABC法检测不同浓度的EGCG（0、25、50、75、100、125mg/L）干预48h后，caspase-3、caspase-8蛋白在Tca8113细胞中的表达差异。 结果：MTT法检测结果显示，不同剂量的EGCG（25、50、75、100、125mg/L）均能抑制口腔鳞癌Tca8113细胞的增殖，其中（50、75、100、125mg/L）与对照组比较差异有统计学意义（p0.05），细胞增值抑制率随着EGCG浓度的增加和作用时间的延长而升高，呈现出明显的时间-剂量效应关系。倒置显微镜下可见对照组的Tca8113细胞呈梭形或多边形贴壁生长，数量密集，轮廓清楚，细胞间连接紧密。加药组的Tca8113细胞随着EGCG浓度的增加，贴壁的细胞数量明显减少，折光性差，部分细胞变圆、皱缩并漂浮于培养基中。AO/EB荧光染色法显示，，不同浓度的EGCG干预Tca8113细胞中出现了典型的凋亡形态学变化，与对照组比较，随着EGCG浓度的增加，凋亡细胞数不断增加。Hochest33258染色显示，不同浓度的EGCG干预Tca8113细胞中出现了典型的凋亡形态学改变如核浓缩、核碎裂等。与对照组比较，随着EGCG浓度的增加，凋亡细胞数不断增加（n=5,p0.01）。分别采用不同浓度EGCG（0、50、75mg/L）处理Tca8113细胞48h后通过流式细胞仪进行细胞周期分析，结果显示EGCG（50、75mg/L）处理组G1期细胞百分数分别为41.997±0.17和65.24±0.632，与对照组（未加药组）比较具有显著性差异（p0.01），表明EGCG诱导Tca8113细胞G1期阻滞。透射电镜下发现与对照组相比，加药组（75mg/L）出现典型的凋亡改变。免疫细胞化学检测结果显示，EGCG能上调Tca8113细胞中caspase-3、caspase-8蛋白的表达，且呈浓度依赖关系。不同浓度的EGCG（0,、25、50、75、100mg/L）干预Tca8113细胞48h，caspase-8蛋白的平均光密度（AOD）值分别为：0.131±0.04、0.177±0.019、0.223±0.06、0.376±0.074、0.571±0.085，各加药组与空白对照组相比差异有统计学意义（p0.01），caspase-3蛋白的平均光密度（AOD）值分别为：0.096±0.072、0.118±0.049、0.197±0.081、0.343±0.033、0.653±0.065，各加药组与空白对照组相比差异有统计学意义（p0.05）。 结论：EGCG能有效地抑制Tca8113细胞的增殖，诱导其凋亡，其机制可能与上调caspase-8、caspase-3的表达有关。
[Abstract]:Aim: to investigate the apoptosis of oral squamous cell carcinoma (Tca8113) cells induced by epigallocathechin-3-gallate EGCG, an epigallocathin-3-gallateEGCG extract from green tea extract. To provide a theoretical basis for EGCG in the prevention and treatment of oral squamous cell carcinoma. Methods: Tca8113 cells were treated with different doses of EGCG 2550U 75100125 mg / L for 24 h, 48 h and 72 h, respectively. The effect of the drug on the growth of Tca8113 cells was observed and the cell proliferation inhibition rate was calculated. Morphologic changes of Tca8113 cells after 48 h treatment with EGCG 050,75100mg / L) .AOP / EB fluorescence staining method was used to detect the morphological changes of Tca8113 cells treated with different dosages of EGCGG 02550mg / L respectively for 48 h. Hochest33258 staining method was used to detect the morphologic changes of Tca8113 cells with different doses of EGCG02550Mg / L (75100125mg / L). The effect on apoptosis of Tca8113 cells. Flow cytometry was used to detect the changes of Tca8113 cell cycle after treatment with EGCG 50 mg / L for 48 h. Transmission electron microscope was used to observe the difference between the drug adding group and the negative control group. The expression of caspase-3 and caspase-8 protein in Tca8113 cells was detected by immunocytochemistry SABC method. Results the cell proliferation of oral squamous cell carcinoma (Tca8113) cells was inhibited by different doses of EGCG2550575100125mg / L, and there was a significant difference between the control group and the control group (P 0.05). The cell proliferation inhibition rate increased with the increase of EGCG concentration and the prolongation of the time of action. There is an obvious time-dose-effect relationship. Under the inverted microscope, the Tca8113 cells in the control group were spindle-shaped or polygonal adherent, dense in number, clear in outline and closely connected between cells. With the increase of EGCG concentration, the number of adherent cells decreased significantly, some cells became round, shrinked and floated in the medium with fluorescence staining. Typical morphological changes of apoptosis appeared in Tca8113 cells treated with different concentrations of EGCG. Compared with the control group, the number of apoptotic cells increased with the increase of EGCG concentration. Hochest33258 staining showed that the number of apoptotic cells increased with the increase of EGCG concentration. Typical apoptotic morphological changes such as nuclear concentration and nuclear fragmentation appeared in Tca8113 cells treated with different concentrations of EGCG. Compared with the control group, the number of apoptotic cells increased with the increase of EGCG concentration. Tca8113 cells were treated with different concentrations of EGCG 50 mg / L for 48 h, and the cell cycle was analyzed by flow cytometry. The results showed that the percentage of G 1 phase cells in EGCG 50 mg / L group was 41.997 卤0.17 and 65.24 卤0.632, respectively, which was significantly different from that in control group (p0.01), indicating that EGCG induced Tca8113 cell G1 phase arrest. Compared with the control group, 75 mg / L of apoptosis was observed under TEM. The results of immunocytochemistry showed that EGCG could up-regulate the expression of caspase-3 and caspase-8 in Tca8113 cells in a concentration-dependent manner. The average optical density of caspase-8 protein was 0.131 卤0.04 卤0.177 卤0.019 卤0.223 卤0.076 卤0.074 卤0.571 卤0.085, respectively. The average optical density of caspase-8 protein was 0.096 卤0.072 卤0.118 卤0.049 卤0.197 卤0.033 卤0.653 卤0.0655.Compared with the blank control group, the average optical density of caspase-3 protein was 0.096 卤0.072 卤0.118 卤0.049 卤0.197 卤0.081 卤0.343 卤0.033 卤0.653 卤0.0655.Compared with the blank control group, the average optical density of caspase-3 protein in each drug group was significantly higher than that in the blank control group. The average optical density of caspase-8 protein was 0.096 卤0.072 卤0.118 卤0.049 卤0.197 卤0.081 卤0.343 卤0.033 卤0.653 卤0.065, respectively. The difference between the two groups was statistically significant (P 0.05). ConclusionEGCG can effectively inhibit the proliferation and induce apoptosis of Tca8113 cells, which may be related to the up-regulation of caspase-8 and caspase-3 expression.
【学位授予单位】：皖南医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.85

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