蟾蜍水提物诱导人肝癌SMMC7721细胞凋亡的初步研究
摘要
目的:观察蟾蜍水提物(Water-soluble extract components of Toad,WECT)对人肝癌细胞株SMMC7721细胞增殖及凋亡情况的影响;检测蟾蜍水提物对SMMC7721细胞株细胞周期的影响,并对其诱导凋亡的相关作用机制进行初步探讨。
方法:以不同浓度的蟾蜍水提物作用于肝癌细胞株SMMC7721,应用噻唑蓝还原法(MTT)检测蟾蜍水提物对肝癌细胞株SMMC7721细胞增殖的抑制作用及细胞毒作用;应用倒置显微镜观察蟾蜍水提物对SMMC7721细胞形态学的影响;Annexin V/PI双染色法检测蟾蜍水提物干预后SMMC7721细胞凋亡及坏死的情况;dUTP缺口末端标记技术,即TUNEL法检测蟾蜍水提物干预后SMMC7721细胞的早期凋亡;流式细胞术分析SMMC7721细胞经药物作用后其周期分布情况的变化。
结果:(1).蟾蜍水提物可以明显抑制细胞分裂增殖,其半数抑制浓度约0.065mg/ml,随着浓度增加诱导细胞凋亡时间缩短,程度增加; (2)蟾蜍水提物能显著抑制SMMC7721细胞生长,生长曲线显示WECT组细胞的增殖速度远慢于对照组,不同时段的WECT组细胞抑制率也明显升高(P﹤0.05);干预后的SMMC7721细胞出现了典型的病理变化,细胞形态由多边形变成圆形,染色质疏松,细胞核溶解,形态学改变具有时间依赖性。(3)细胞凋亡与WECT程浓度和时间依赖关系,随着浓度的增加及时间的延长细胞凋亡比例明显增加,与阴性对照有显著差异(P﹤0.05)。(4)流式细胞仪测定显示细胞周期发生明显改变,细胞分裂阻滞在S期,与阴性对照组比较有显著差异性(P﹤0.05)。
结论:1.0.008mg/mL及以上浓度蟾蜍水提物对人肝癌SMMC7721细胞具有显著细胞毒作用,形态学和DNA片段化检测证实蟾蜍水提物诱导的细胞死亡以凋亡为主; 2.蟾蜍水提物对肝癌SMMC7721细胞增殖抑制作用呈浓度、时间依赖性,24、48和72小时的半数抑制浓度分别为:0.214mg/mL、0.065mg/mL、0.023 mg/mL。3 .蟾蜍水提物可以诱导SMMC7721细胞凋亡,且呈现明显的浓度、时间依赖性。4.蟾蜍水提物可以抑制SMMC7721细胞生长,并将细胞周期阻滞于S期,诱导肝癌细胞周期S期阻滞可能是蟾蜍水提物抑制肝癌细胞增殖的机制之一。
Objective:To observe the proliferation and apotosis of human hepatoma cell strain SMMC7721 induced by Water-soluble extract components of Toad and detection the effect of cell cycle on SMMC7721 cells by Water-soluble extract components of Toad and preliminaryly investigate the related mechanism of the apoptosis.
Methods:The SMMC7721 cells were treated with Water-soluble extract components of Toad of different concentrations. The growth inhibition and cytotoxicity of Water-soluble extract components of Toad on SMMC7721 cells was measured by MTT reduction law assay; The change of morphologic of SMMC7721 cells which were intervented by Water-soluble extract components of Toad was observed by the inverted microscopy. Flow cytometry and Annexin-V/PI was used to study the apoptosis and Necrosis; Cell cycle distribution was analyzed with flow
cytometry.
Results:(1) The water-soluble extract components of Toad could remarkably inhibited the cell growth, the IC50 value of Water-soluble extract components of Toad for cells was 0.065mg/mL and the effect enhanced with it’s concentration.(2) Water-soluble extract components of Toad can significantly inhibit the growth of SMMC7721 cells, the growth curve shows that the proliferation of the cells which were dealed with the water-soluble extract components of Toad was far slower than the control group. Different time slots cells also significantly increased inhibition rate(P﹤0.01﹚ After the intervention, SMMC7721 cells performance a typical pathological changes and the morphological changes with time-dependent. Cell changed from polygon into round. Chromatin looseness and karylysis were observed. (3) The Apoptosis have dependency relation to the drug density and drug action time, the percentage of apoptosis increase along with the density increase and the time extension. There was a significant difference(P﹤0.01)between drug group and control group in apoptosis rate. (4) There is a remarkably change of cell cycle distribution which was analyzed with flow cytometry , Treatment of cells with Water-soluble extract components of Toad induced arrest in S phase of cell cycle progression.
Conclusion:1. The Water-soluble extract components of Toad at concentration of 0.004mg/ml or higher could have a remarkable cytotoxic effect on human hepatoma cells SMMC7721. Water-soluble extract components of Toad could efficiently induce apoptosis and necrosis in SMMC 7721 cells. 2. Water-soluble extract components of Toad suppresses the multiplication of SMMC7721 cells in dose and time - dependent manner. The IC50 value of Water-soluble extract components of Toad for cells was 0.214mg/mL、0.065mg/mL and 0.023 mg/mL which was treated distinctive for 24、48 and 72 hours.3. Water-soluble extract components of Toad induced apoptosis of SMMC7721 cells. And it assumes the time and the density dependence. 4. Water-soluble extract components of Toad could inhibit SMMC7721 cell growth and arrest cells in S phase of cell cycle,which is possibly one of the mechanisms for anticancer of Water-soluble extract components of Toad.
方法:以不同浓度的蟾蜍水提物作用于肝癌细胞株SMMC7721,应用噻唑蓝还原法(MTT)检测蟾蜍水提物对肝癌细胞株SMMC7721细胞增殖的抑制作用及细胞毒作用;应用倒置显微镜观察蟾蜍水提物对SMMC7721细胞形态学的影响;Annexin V/PI双染色法检测蟾蜍水提物干预后SMMC7721细胞凋亡及坏死的情况;dUTP缺口末端标记技术,即TUNEL法检测蟾蜍水提物干预后SMMC7721细胞的早期凋亡;流式细胞术分析SMMC7721细胞经药物作用后其周期分布情况的变化。
结果:(1).蟾蜍水提物可以明显抑制细胞分裂增殖,其半数抑制浓度约0.065mg/ml,随着浓度增加诱导细胞凋亡时间缩短,程度增加; (2)蟾蜍水提物能显著抑制SMMC7721细胞生长,生长曲线显示WECT组细胞的增殖速度远慢于对照组,不同时段的WECT组细胞抑制率也明显升高(P﹤0.05);干预后的SMMC7721细胞出现了典型的病理变化,细胞形态由多边形变成圆形,染色质疏松,细胞核溶解,形态学改变具有时间依赖性。(3)细胞凋亡与WECT程浓度和时间依赖关系,随着浓度的增加及时间的延长细胞凋亡比例明显增加,与阴性对照有显著差异(P﹤0.05)。(4)流式细胞仪测定显示细胞周期发生明显改变,细胞分裂阻滞在S期,与阴性对照组比较有显著差异性(P﹤0.05)。
结论:1.0.008mg/mL及以上浓度蟾蜍水提物对人肝癌SMMC7721细胞具有显著细胞毒作用,形态学和DNA片段化检测证实蟾蜍水提物诱导的细胞死亡以凋亡为主; 2.蟾蜍水提物对肝癌SMMC7721细胞增殖抑制作用呈浓度、时间依赖性,24、48和72小时的半数抑制浓度分别为:0.214mg/mL、0.065mg/mL、0.023 mg/mL。3 .蟾蜍水提物可以诱导SMMC7721细胞凋亡,且呈现明显的浓度、时间依赖性。4.蟾蜍水提物可以抑制SMMC7721细胞生长,并将细胞周期阻滞于S期,诱导肝癌细胞周期S期阻滞可能是蟾蜍水提物抑制肝癌细胞增殖的机制之一。
Objective:To observe the proliferation and apotosis of human hepatoma cell strain SMMC7721 induced by Water-soluble extract components of Toad and detection the effect of cell cycle on SMMC7721 cells by Water-soluble extract components of Toad and preliminaryly investigate the related mechanism of the apoptosis.
Methods:The SMMC7721 cells were treated with Water-soluble extract components of Toad of different concentrations. The growth inhibition and cytotoxicity of Water-soluble extract components of Toad on SMMC7721 cells was measured by MTT reduction law assay; The change of morphologic of SMMC7721 cells which were intervented by Water-soluble extract components of Toad was observed by the inverted microscopy. Flow cytometry and Annexin-V/PI was used to study the apoptosis and Necrosis; Cell cycle distribution was analyzed with flow
cytometry.
Results:(1) The water-soluble extract components of Toad could remarkably inhibited the cell growth, the IC50 value of Water-soluble extract components of Toad for cells was 0.065mg/mL and the effect enhanced with it’s concentration.(2) Water-soluble extract components of Toad can significantly inhibit the growth of SMMC7721 cells, the growth curve shows that the proliferation of the cells which were dealed with the water-soluble extract components of Toad was far slower than the control group. Different time slots cells also significantly increased inhibition rate(P﹤0.01﹚ After the intervention, SMMC7721 cells performance a typical pathological changes and the morphological changes with time-dependent. Cell changed from polygon into round. Chromatin looseness and karylysis were observed. (3) The Apoptosis have dependency relation to the drug density and drug action time, the percentage of apoptosis increase along with the density increase and the time extension. There was a significant difference(P﹤0.01)between drug group and control group in apoptosis rate. (4) There is a remarkably change of cell cycle distribution which was analyzed with flow cytometry , Treatment of cells with Water-soluble extract components of Toad induced arrest in S phase of cell cycle progression.
Conclusion:1. The Water-soluble extract components of Toad at concentration of 0.004mg/ml or higher could have a remarkable cytotoxic effect on human hepatoma cells SMMC7721. Water-soluble extract components of Toad could efficiently induce apoptosis and necrosis in SMMC 7721 cells. 2. Water-soluble extract components of Toad suppresses the multiplication of SMMC7721 cells in dose and time - dependent manner. The IC50 value of Water-soluble extract components of Toad for cells was 0.214mg/mL、0.065mg/mL and 0.023 mg/mL which was treated distinctive for 24、48 and 72 hours.3. Water-soluble extract components of Toad induced apoptosis of SMMC7721 cells. And it assumes the time and the density dependence. 4. Water-soluble extract components of Toad could inhibit SMMC7721 cell growth and arrest cells in S phase of cell cycle,which is possibly one of the mechanisms for anticancer of Water-soluble extract components of Toad.
引文
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