温化胶囊对人肝癌细胞株Bel-7402凋亡和代谢的影响
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摘要
目的:研究中药复方制剂温化胶囊对人肝癌细胞株Bel-7402的凋亡和代谢的影响,本次研究力求在原有研究成果的基础上,通过对人肝癌Bel—7402细胞株的体外培养,来进一步观察温化胶囊对于肿瘤细胞的影响,。
方法:实验共分四部分。第一部分:对人肝癌细胞株Bel-7402进行体外细胞培养,分设空白对照组、150μg/ml温化胶囊组及750μg/ml温化胶囊组三组,使用流式细胞仪分析温化胶囊对人肝癌细胞株Bel-7402凋亡的影响。利用倒置相差显微镜观察细胞株加药前后的形态学变化。第二部分:速率法检测温化胶囊对Bel-7402细胞培养上清液中乳酸脱氢酶(LDH)水平的影响。第三部分:应用酶联免疫吸附剂测定(enzyme linked immunosorbent assay,ELISA)的检测方法检测温化胶囊对Bel-7402细胞培养上清液中血管内皮细胞生长因子(VEGF)水平的影响。第四部分:应用ELISA的检测方法检测温化胶囊对Bel-7402细胞培养上清液中转化生长因子β1(TGF-β1)水平的影响。
结果:流式细胞仪检测显示:与对照组相比较,温化胶囊能够提高Bel-7402细胞的凋亡率,其作用的强度与药物的剂量呈正相关;细胞形态学观察发现,加入温化胶囊后细胞质内出现了空泡样结构,细胞变小、或变细变长,或变圆,部分细胞脱落漂浮于培养基中,且随着药物作用时间的延长和药物浓度的增加,空泡样结构的数量亦呈进行性增加,并可融合成大泡,其漂浮细胞也增多;对LDH细胞因子的检测表明,温化胶囊高剂量组(750μg/ml)的LDH水平明显高于对照组(P＜0.05),但温化胶囊高剂量组(750μg/ml)和温化胶囊低剂量组(150μg/ml)、温化胶囊低剂量组(150μg/ml)和对照组相比较,二者均没有显著性差异(P＞0.05),本次研究中温化胶囊高剂量组(750μg/ml)的LDH水平明显高于对照组,有统计学差异,反应出高剂量组的Bel-7402细胞细胞膜的受损程度高于对照组,即温化胶囊对此种细胞株的细胞毒性作用强于对照组。应用ELISA方法检测VEGF和TGF-β1两因子的水平,温化胶囊高剂量组(750μg/ml)与对照组相比,TGF-β1上调,有显著性差异(P＜0.05),温化胶囊低剂量组(150μg/ml)与对照组比较未见显著性差异(P＞0.05),而温化胶囊对VEGF的作用不明显,本次实验中对照组、温化胶囊低剂量组(150μg/ml)与温化胶囊高剂量组(750μg/ml)三组相比较,P＞0.05,均无显著性差异。
结论:温化胶囊立足《内经》有关肿瘤的论述,通过温阳散寒与化痰活血从而发挥温阳化气散结的作用。全方仅五味药,主要药物为附子、贝母。通过温化胶囊对肝癌细胞系Bel-7402细胞的作用观察,我们发现温化胶囊的诱导凋亡和质膜破坏作用是肯定的。经过温化胶囊处理后细胞形态发生明显变化,流式细胞仪检测提示细胞凋亡率增加,细胞培养上清中的LDH浓度升高,上调细胞分泌的TGF-β1的表达,发挥了诱导凋亡、抑制肿瘤的作用。而对VEGF的作用不明显。
Objective:To investigate the effect of apoptosis and metabolism of a Chinese medical mixture,WenHua capsules,on Bel-7402 cells.
Methods:The experiment was divided into four parts.The first part,Bel-7402 cells were cultured in vitro.To set up three observation groups,that is negative control group,the group being exposed to 150μg/ml concentration of WenHua capsules,the group being exposed to 750μg/ml concentration of WenHua capsules.Apoptosis of Bel-7402 was detected by flow cytometry.The change of cell morphology after being exposed to WenHua capsules was observed by using inverted phase contrast microscope.The second part,the secretion of lactate dehydrogenase was detected by velocimetry.The third part,the secretion of VEGF was detected by enzyme linked immunosorbent assay(ELISA).The fourth part,the secretion of transforming growth factorβ1(TGF-β1)was detected by ELISA.
Results:The percentage of apoptosis cells increased on WenHua capsules-treated cells,and the relationship between the percentage and the concentration of WenHua capsules was positive correlation.After being exposed to WenHua capsules,the cells were changed into smaller,or longer,or more round.Some vacuole-like structures could be observed in intracytoplasm of some cells.In the mediam,some cells floated. The higher concentration of WenHua capsules,the more obvious of this kind of phenomenon.The lactate dehydrogenase secretion increased after being exposed to 750μg/ml concentration of WenHua capsules(P＜0.05),but the difference between the group treated with 150μg/ml concentration of WenHua capsules and the negative control group was not obvious(P＞0.05).The TGF-β1 secretion in the group being exposed to 750μg/ml concentration of WenHua capsules increased(P＜0.05)while the change of the VEGF secretion among the three groups was not obvious(P＞0.05).
Conclusion:WenHua capsules can induce the apoptosis of Bel-7402 cell.It has strong cytotoxicity to Bel-7402 cells,it can make cells hydropic degeneration,increase the secretion of lactate dehydrogenase and TGF-β1.The effect on the secretion of VEGF was not obvious.
方法:实验共分四部分。第一部分:对人肝癌细胞株Bel-7402进行体外细胞培养,分设空白对照组、150μg/ml温化胶囊组及750μg/ml温化胶囊组三组,使用流式细胞仪分析温化胶囊对人肝癌细胞株Bel-7402凋亡的影响。利用倒置相差显微镜观察细胞株加药前后的形态学变化。第二部分:速率法检测温化胶囊对Bel-7402细胞培养上清液中乳酸脱氢酶(LDH)水平的影响。第三部分:应用酶联免疫吸附剂测定(enzyme linked immunosorbent assay,ELISA)的检测方法检测温化胶囊对Bel-7402细胞培养上清液中血管内皮细胞生长因子(VEGF)水平的影响。第四部分:应用ELISA的检测方法检测温化胶囊对Bel-7402细胞培养上清液中转化生长因子β1(TGF-β1)水平的影响。
结果:流式细胞仪检测显示:与对照组相比较,温化胶囊能够提高Bel-7402细胞的凋亡率,其作用的强度与药物的剂量呈正相关;细胞形态学观察发现,加入温化胶囊后细胞质内出现了空泡样结构,细胞变小、或变细变长,或变圆,部分细胞脱落漂浮于培养基中,且随着药物作用时间的延长和药物浓度的增加,空泡样结构的数量亦呈进行性增加,并可融合成大泡,其漂浮细胞也增多;对LDH细胞因子的检测表明,温化胶囊高剂量组(750μg/ml)的LDH水平明显高于对照组(P＜0.05),但温化胶囊高剂量组(750μg/ml)和温化胶囊低剂量组(150μg/ml)、温化胶囊低剂量组(150μg/ml)和对照组相比较,二者均没有显著性差异(P＞0.05),本次研究中温化胶囊高剂量组(750μg/ml)的LDH水平明显高于对照组,有统计学差异,反应出高剂量组的Bel-7402细胞细胞膜的受损程度高于对照组,即温化胶囊对此种细胞株的细胞毒性作用强于对照组。应用ELISA方法检测VEGF和TGF-β1两因子的水平,温化胶囊高剂量组(750μg/ml)与对照组相比,TGF-β1上调,有显著性差异(P＜0.05),温化胶囊低剂量组(150μg/ml)与对照组比较未见显著性差异(P＞0.05),而温化胶囊对VEGF的作用不明显,本次实验中对照组、温化胶囊低剂量组(150μg/ml)与温化胶囊高剂量组(750μg/ml)三组相比较,P＞0.05,均无显著性差异。
结论:温化胶囊立足《内经》有关肿瘤的论述,通过温阳散寒与化痰活血从而发挥温阳化气散结的作用。全方仅五味药,主要药物为附子、贝母。通过温化胶囊对肝癌细胞系Bel-7402细胞的作用观察,我们发现温化胶囊的诱导凋亡和质膜破坏作用是肯定的。经过温化胶囊处理后细胞形态发生明显变化,流式细胞仪检测提示细胞凋亡率增加,细胞培养上清中的LDH浓度升高,上调细胞分泌的TGF-β1的表达,发挥了诱导凋亡、抑制肿瘤的作用。而对VEGF的作用不明显。
Objective:To investigate the effect of apoptosis and metabolism of a Chinese medical mixture,WenHua capsules,on Bel-7402 cells.
Methods:The experiment was divided into four parts.The first part,Bel-7402 cells were cultured in vitro.To set up three observation groups,that is negative control group,the group being exposed to 150μg/ml concentration of WenHua capsules,the group being exposed to 750μg/ml concentration of WenHua capsules.Apoptosis of Bel-7402 was detected by flow cytometry.The change of cell morphology after being exposed to WenHua capsules was observed by using inverted phase contrast microscope.The second part,the secretion of lactate dehydrogenase was detected by velocimetry.The third part,the secretion of VEGF was detected by enzyme linked immunosorbent assay(ELISA).The fourth part,the secretion of transforming growth factorβ1(TGF-β1)was detected by ELISA.
Results:The percentage of apoptosis cells increased on WenHua capsules-treated cells,and the relationship between the percentage and the concentration of WenHua capsules was positive correlation.After being exposed to WenHua capsules,the cells were changed into smaller,or longer,or more round.Some vacuole-like structures could be observed in intracytoplasm of some cells.In the mediam,some cells floated. The higher concentration of WenHua capsules,the more obvious of this kind of phenomenon.The lactate dehydrogenase secretion increased after being exposed to 750μg/ml concentration of WenHua capsules(P＜0.05),but the difference between the group treated with 150μg/ml concentration of WenHua capsules and the negative control group was not obvious(P＞0.05).The TGF-β1 secretion in the group being exposed to 750μg/ml concentration of WenHua capsules increased(P＜0.05)while the change of the VEGF secretion among the three groups was not obvious(P＞0.05).
Conclusion:WenHua capsules can induce the apoptosis of Bel-7402 cell.It has strong cytotoxicity to Bel-7402 cells,it can make cells hydropic degeneration,increase the secretion of lactate dehydrogenase and TGF-β1.The effect on the secretion of VEGF was not obvious.
引文
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