大黄素对人结肠癌HT-29细胞体外增殖的抑制作用

Inhibitory action of emodin on proliferation of colon cancer cell HT-29 in vitro

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 discusses the inhibitory action of emodin on proliferation of HT-29 cell in vitro. Methods HT-29 cells in logarithmic growth phase were incubated with different concentrations of emodin （12.5, 25, 50, 100 μM） and randomized into five groups： control group and four experimental groups. Six duplicated holes in each group, cultivated for 24 h, 48 h and 72 h respectively at 37 ℃, in 5% CO2 incubator. The cell activity was detected by MTT, and the quantity of cells was observed ; the apoptosis rate and cell cycle distribution were detected by FCM, and the activity of Caspase-3 was detected by spectrophotometry. Results （1） The result of MTT showed： after 24h, compared with control group, there was significant difference in inhibition ratio between 50 μM and 100μM emodin group （P 〈 0.05 ） , no significant difference was found in 12.5 μM and 25 μM emodin group （P 〉 0.05 ）. Compared with control group, after 48 h and 72 h, there were significant difference in inhibition ratio between control group and the four emodin groups （P 〈 0.05）. The higher of the emodin, the higher inhibitory rate of HT-29 in different groups of the same processing time. Compared with the 48 h and 72 h, the inhibition ratio at 24 h was increased significantly among the emodin groups （ P 〈 0.05 ） ; in emodin groups, there was significant difference in inhibition ratio between 72 h and 48 h （ P 〈 0.05 ）. （2） inverted microscope showed ： HT-29 cells usually grow round-clustering, after intervened by emodin, the volume was reduced, space was narrowed and the count of the cells were reduced. （3） the FCM showed ： cell ratio at G2/M phase in control group was higher than that in emodin groups significantly （ P 〈0.05 ） ; cell ratio at G0/G1 phase in control group was higher than that in 25 μM, 50 μM and 100 μM emodin groups significantly （P 〈0.05 ） ; no significant difference was found in cell ratio at G0/G1 phase between control group and 12.5 μM emodin group （P 〉 0.05）. The cell ratio at S phase in control group was lower than that in emodin groups significantly （ P 〈 0.05 ）, it showed that emodin retarded HT-29 cells at S phrase. Compared with control group, the apoptosis rate of HT-29 cells was increased in four emodin groups significantly （P 〈 0.05 ）, it showed that, emodin promoted the apoptosis of HT-29 cells. （4） Compared with control group, the activity of caspase-3 protein in emodin groups was increased significantly （ P 〈 0.05 ）. Conclusion Emodin can inhibit the proliferations of human colon cancer cell lines HT-29 in vitro in a dose-dependent and time-dependent manner; it can retard the cell cycle and induce cell apoptosis by activating Caspase-3.