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目的观察大黄素对人结肠癌细胞株HT-29体外增殖的抑制作用。方法取对数生长期的HT-29细胞,按每孔5×103个细胞接种于96孔板,贴壁24 h后换液,分为对照组和实验组(分别含有12.5μM、25μM、50μM、100μM大黄素),共5组。每组均设6个复孔,于37°C、5%CO2孵箱中分别培养24 h、48 h和72 h。采用MTT比色法检测细胞活力;倒置显微镜显像观察细胞数量变化;流式细胞仪检测细胞凋亡率及周期分布;分光光度法检测Caspase-3酶蛋白活性。结果①MTT比色法显示,处理24 h时,与对照组比较,大黄素50μM、100μM组细胞抑制率差异有统计学意义(P<0.05),而大黄素12.5μM、25μM组抑制率差异无统计学意义(P>0.05)。与对照组比较,处理48 h及72 h后大黄素12.5μM、25μM、50μM、100μM组的细胞抑制率差异有统计学意义(P<0.05);大黄素各浓度组处理48 h及72 h后的抑制率与同浓度组处理24 h比较,抑制率升高,差异有统计学意义(P<0.05);大黄素各浓度组处理72 h后的抑制率与同浓度组处理48 h比较抑制率升高,差异有统计学意义(P<0.05)。②倒置显微镜观测显示,对照组多为类圆形成团生长,经各浓度大黄素处理后,细胞体积缩小、间隙变大,细胞数减少。③流式细胞仪检测显示,对照组G2/M期细胞比率明显高于大黄素各浓度组,差异有统计学意义(P<0.05);对照组G0/G1期细胞比率明显高于大黄素25μM、50μM、100μM浓度组,差异有统计学意义(P<0.05);对照组与12.5μM大黄素组G0/G1期细胞比率差异无统计学意义(P>0.05);对照组S期细胞比率明显低于大黄素各浓度组,差异有统计学意义(P<0.05)。与对照组比较,大黄素12.5μM、25μM、50μM、100μM组HT-29细胞凋亡率增加,差异有统计学意义(P<0.05)。④与对照组比较,大黄素各浓度组Caspase-3酶蛋白活性显著增高,差异有统计学意义(P<0.05)。结论大黄素抑制人结肠癌细胞株HT-29体外增殖,其作用呈浓度、时间依赖性;大黄素能够阻滞HT-29细胞周期,并激活Caspase-3诱导细胞凋亡。
Objective To observe the inhibitory effect of emodin on the proliferation of human colon cancer cell line HT-29 in vitro. Methods HT-29 cells in logarithmic growth phase were seeded in 96-well plates at a density of 5 × 10 3 cells per well and were transplanted for 24 h. The HT-29 cells were divided into control group and experimental group (containing 12.5 μM, 25 μM, 50 μM , 100 μM emodin), a total of 5 groups. Six replicate wells were set up in each group and cultured in 37 ° C, 5% CO2 incubator for 24 h, 48 h and 72 h, respectively. Cell viability was detected by MTT colorimetric assay. The number of cells was observed by inverted microscope. The apoptosis rate and period distribution were detected by flow cytometry. The protein activity of Caspase-3 was detected by spectrophotometry. Results ① MTT assay showed that the inhibitory rates of emodin 50μM and 100μM groups were significantly lower than those of the control group at 24 h (P <0.05), but there was no statistical difference between the emodin groups at 12.5μM and 25μM Significance (P> 0.05). Compared with the control group, the inhibitory rates of emodin 12.5μM, 25μM, 50μM and 100μM after 48 h and 72 h treatment were significantly different (P <0.05). After treated with emodin for 48 h and 72 h (P <0.05). Compared with the same concentration group for 24 h, the inhibition rate increased significantly (P <0.05) Increased, the difference was statistically significant (P <0.05). ② Inverted microscope observation showed that the control group mostly grew in round clusters. After being treated with various concentrations of emodin, the cell volume was reduced, the gap became larger and the number of cells decreased. Flow cytometry showed that the cell ratio of G2 / M phase in control group was significantly higher than that of emodin group (P <0.05), and the percentage of cells in G0 / G1 phase in control group was significantly higher than that of emodin 25μM , 50μM and 100μM (P <0.05). There was no significant difference in G0 / G1 phase cell ratio between the control group and 12.5μM emodin group (P> 0.05). The S phase cell ratio in the control group was significantly Lower than emodin concentration, the difference was statistically significant (P <0.05). Compared with the control group, the apoptotic rates of HT-29 cells in emodin 12.5μM, 25μM, 50μM and 100μM group increased significantly (P <0.05). ④ Compared with the control group, the activity of Caspase-3 protein in each concentration of emodin significantly increased, with statistical significance (P <0.05). Conclusion Emodin can inhibit the proliferation of human colon cancer cell line HT-29 in vitro in a time-and concentration-dependent manner. Emodin can block the cell cycle of HT-29 cells and activate Caspase-3 to induce cell apoptosis.