复方斑蝥的抗肝癌作用研究
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摘要
目的探讨复方斑蝥对肝癌的作用效果和作用机制。
方法用血清药理学方法制备复方斑蝥血清并处理人肝癌Bel-7402细胞, MTT方法观察其对人肝癌Bel-7402细胞增殖的抑制作用, RT-PCR技术检测其对c-Myc,P53基因表达的影响,流式细胞仪检测含药血清对细胞周期的作用;使用鼠肝癌细胞H22,建立肝腹水模型,观察复方斑蝥对荷腹水瘤小鼠生存质量及生存期的影响。
结果复方斑蝥含药血清能够显著抑制人肝癌Bel-7402细胞的增殖。通过MTT实验观察不同含药浓度血清组对人肝癌Bel-7402细胞作用24h、48h、72h,复方斑蝥含药血清对人肝癌Bel-7402细胞的抑制作用呈现时间-浓度的依赖性。低浓度、中浓度、高浓度含药血清作用24h后对人肝癌Bel-7402细胞增殖的抑制率分别为:6%、12%、13%;作用48h后对人肝癌Bel-7402细胞增殖的抑制率分别为:13%、15%、27%;作用72h后对人肝癌Bel-7402细胞增殖的抑制率分别为:15%、32%、32%;与对照组血清相比较,含药血清对人肝癌Bel-7402细胞增殖抑制作用存在显著性差异(p<0.05)。通过含药血清对人肝癌Bel-7402细胞作用48h,RT-PCR实验检测到含药血清能够下调c-Myc,上调P53基因的表达;流式细胞仪观察不同浓度药物的含药血清将细胞阻滞在G2M期。其中中、高浓度药物组血清作用三个时间对后,与对照组相比较存在显著差异性(p<0.05)。复方斑蝥治疗肝腹水模型小鼠,对其生存期延长效果不明显,但对其生存质量(如毛色、活动能力、对环境的敏感性等)有明显提升作用。
结论复方斑蝥能够抑制人肝癌细胞的增殖,其作用机制可能是通过影响P53、c-Myc基因表达,进而将细胞阻滞在G2M期来实现。体内实验显示复方斑蝥可能提高荷瘤小鼠的免疫力,从而改善荷瘤小鼠生存质量。血清药理学与分子生物学相结合能够有效地展示与药物作用相关的分子,可以作为研究复方药理的一个初步技术平台。
Objective To investigate the antitumor effect of compound mylabris on the hepatocellular carcinoma and its possible mechanism.
Methods The compound mylabris serum was prepared based on serum pharmacology and was used to treat human hepatocarcinoma cell line bel-7402. Different concentrations of compound mylabris serum were used to treat bel-7402 cell for 24 h, 48 h, 78 h, and their inhibitory effect were detected by MTT assay. RT-polymerase chain reaction (RT-PCR) was used to evaluate c-Myc and p53 mRNA expressions in human Hepatocarcinoma cell bel-7402. Flow cytometry was used to detect the effect of compound mylabris serum on cell cycle of bel-7402. The H22 hepatoma ascites bearing mouse model was set up, and was used to investigate the effect of the compound mylabris on the life time of the model mouse.
Results Compound mylabris serum could inhibit remarkably the proliferation of human hepatocarcinoma cell line bel-7402. The MTT results showed that inhibitory effect of compound mylabris serum on bel-7402 was time and concentration dependent. The inhibitory rates of the groups of low, middle and high concentration for 24 hours were 6%, 12%, 13%, respectively; while for the groups of 48 hours and 72 hours, they were 13%, 15%, 27% and 15%, 32%, 32%, respectively. In comparison with the control group, the groups of compound mylabris serum showed significant inhibitory effect (p < 0.05). The results of RT–PCR showed that the compound mylabris can downregulate c-Myc gene, while upregulate p53 gene. The flow cytometry showed that cell cycles of bel-7402 were blocked into the G2M phase after being treated by compound mylabris serum with middle and high concentration for 24, 48 and 72 hours (p < 0.05). The compound mylabris can not significantly lengthen the life span of H22 hepatoma ascites bearing mouse model, but it can remarkably improve their life quality, such as hair color, moving ability and sensitivity to the environment.
Conclusion Compound mylabris can inhibit the proliferation of human hepatocarcinoma cell. This may be achieved by affecting the expressions of P53 and c-Myc gene, and thereby blocking the cell cycle into the G2M phase. Compound mylabris may improve the immunocompetence of the tumor bearing mouse and thus better its life quality. Serum pharmacology in combination with molecular biology can effectively disclose target molecules reacting with the drug, and therefore can be used as a preliminary technology platform for compound pharmacology.
方法用血清药理学方法制备复方斑蝥血清并处理人肝癌Bel-7402细胞, MTT方法观察其对人肝癌Bel-7402细胞增殖的抑制作用, RT-PCR技术检测其对c-Myc,P53基因表达的影响,流式细胞仪检测含药血清对细胞周期的作用;使用鼠肝癌细胞H22,建立肝腹水模型,观察复方斑蝥对荷腹水瘤小鼠生存质量及生存期的影响。
结果复方斑蝥含药血清能够显著抑制人肝癌Bel-7402细胞的增殖。通过MTT实验观察不同含药浓度血清组对人肝癌Bel-7402细胞作用24h、48h、72h,复方斑蝥含药血清对人肝癌Bel-7402细胞的抑制作用呈现时间-浓度的依赖性。低浓度、中浓度、高浓度含药血清作用24h后对人肝癌Bel-7402细胞增殖的抑制率分别为:6%、12%、13%;作用48h后对人肝癌Bel-7402细胞增殖的抑制率分别为:13%、15%、27%;作用72h后对人肝癌Bel-7402细胞增殖的抑制率分别为:15%、32%、32%;与对照组血清相比较,含药血清对人肝癌Bel-7402细胞增殖抑制作用存在显著性差异(p<0.05)。通过含药血清对人肝癌Bel-7402细胞作用48h,RT-PCR实验检测到含药血清能够下调c-Myc,上调P53基因的表达;流式细胞仪观察不同浓度药物的含药血清将细胞阻滞在G2M期。其中中、高浓度药物组血清作用三个时间对后,与对照组相比较存在显著差异性(p<0.05)。复方斑蝥治疗肝腹水模型小鼠,对其生存期延长效果不明显,但对其生存质量(如毛色、活动能力、对环境的敏感性等)有明显提升作用。
结论复方斑蝥能够抑制人肝癌细胞的增殖,其作用机制可能是通过影响P53、c-Myc基因表达,进而将细胞阻滞在G2M期来实现。体内实验显示复方斑蝥可能提高荷瘤小鼠的免疫力,从而改善荷瘤小鼠生存质量。血清药理学与分子生物学相结合能够有效地展示与药物作用相关的分子,可以作为研究复方药理的一个初步技术平台。
Objective To investigate the antitumor effect of compound mylabris on the hepatocellular carcinoma and its possible mechanism.
Methods The compound mylabris serum was prepared based on serum pharmacology and was used to treat human hepatocarcinoma cell line bel-7402. Different concentrations of compound mylabris serum were used to treat bel-7402 cell for 24 h, 48 h, 78 h, and their inhibitory effect were detected by MTT assay. RT-polymerase chain reaction (RT-PCR) was used to evaluate c-Myc and p53 mRNA expressions in human Hepatocarcinoma cell bel-7402. Flow cytometry was used to detect the effect of compound mylabris serum on cell cycle of bel-7402. The H22 hepatoma ascites bearing mouse model was set up, and was used to investigate the effect of the compound mylabris on the life time of the model mouse.
Results Compound mylabris serum could inhibit remarkably the proliferation of human hepatocarcinoma cell line bel-7402. The MTT results showed that inhibitory effect of compound mylabris serum on bel-7402 was time and concentration dependent. The inhibitory rates of the groups of low, middle and high concentration for 24 hours were 6%, 12%, 13%, respectively; while for the groups of 48 hours and 72 hours, they were 13%, 15%, 27% and 15%, 32%, 32%, respectively. In comparison with the control group, the groups of compound mylabris serum showed significant inhibitory effect (p < 0.05). The results of RT–PCR showed that the compound mylabris can downregulate c-Myc gene, while upregulate p53 gene. The flow cytometry showed that cell cycles of bel-7402 were blocked into the G2M phase after being treated by compound mylabris serum with middle and high concentration for 24, 48 and 72 hours (p < 0.05). The compound mylabris can not significantly lengthen the life span of H22 hepatoma ascites bearing mouse model, but it can remarkably improve their life quality, such as hair color, moving ability and sensitivity to the environment.
Conclusion Compound mylabris can inhibit the proliferation of human hepatocarcinoma cell. This may be achieved by affecting the expressions of P53 and c-Myc gene, and thereby blocking the cell cycle into the G2M phase. Compound mylabris may improve the immunocompetence of the tumor bearing mouse and thus better its life quality. Serum pharmacology in combination with molecular biology can effectively disclose target molecules reacting with the drug, and therefore can be used as a preliminary technology platform for compound pharmacology.
引文
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