复方苦参注射液及其与奥沙利铂和恩度联合抑制肝癌细胞增殖及诱导凋亡的研究
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摘要
目的:通过体外实验观察复方苦参注射液及其与第三代铂类化疗药物奥沙利铂和抗肿瘤血管生成药物重组人血管内皮抑制素(恩度)联合作用于人肝癌细胞株的效果,并探讨其相关的作用机制,为中药与化疗及抗肿瘤血管生成治疗联合应用提供理论依据。
方法:将不同浓度的复方苦参注射液、奥沙利铂、恩度单药以及联合作用于体外培养的人肝癌细胞株SMMC-7721,使用不同的技术检测细胞生长状况:(1)MTT法分析不同浓度和作用时间的药物对细胞增殖的抑制效果;(2)倒置显微镜观察细胞的形态学变化;(3)荧光显微镜观察细胞凋亡的形态变化特征;(4)流式细胞术检测细胞周期的分布改变;(5)原位末端标记法(TUNEL)检测凋亡细胞的细胞核着色情况;(6)免疫细胞化学法检测细胞凋亡相关蛋白Survivin、Bcl-2、Caspase-3的表达。
结果:(1)复方苦参注射液对肝癌SMMC-7721细胞有抑制增殖的作用,且该作用具有时间、剂量依赖性;50μl/ml的复方苦参注射液与1μg/ml的奥沙利铂联合作用48h后表现出协同作用,但与恩度联合未见明显增强;(2)光镜下观察,药物作用后细胞体积变小,逐渐变圆,细胞间接触变松;(3)荧光显微镜下观察,细胞发生了明显的早期凋亡和晚期凋亡,联合奥沙利铂组凋亡细胞比例显著升高;(4)流式细胞术检测显示药物组G_0/G_1期细胞比例升高,S期比例下降,细胞被阻滞于G_0/G_1期;(5)原位末端标记法(TUNEL)检测到药物作用后,凋亡细胞的细胞核被染成深褐色,当联合奥沙利铂后凋亡指数显著升高,而联合恩度未见明显变化;(6)免疫细胞化学法显示药物作用后,SMMC-7721细胞Survivin、Bcl-2蛋白的表达较对照组减弱(P＜0.01),Caspase-3的表达增高(P＜0.01),当联合奥沙利铂后,这种作用得到显著加强(P＜0.01),而联合恩度未见明显变化。
结论:复方苦参注射液对肝癌SMMC-7721细胞有抑制增殖的作用,该作用可能是通过将细胞阻滞于G_0/G_1期,同时下调Survivin、Bcl-2的表达,上调Caspase-3的表达而诱导细胞凋亡来实现的。当复方苦参注射液与奥沙利铂联合后,其抑制增殖和诱导凋亡的作用显著增强,表现出协同作用;而当联合恩度时,其作用未见明显增强。本研究为临床探索优化的中西医结合治疗原发性肝癌的联合方案提供了实验依据。
Objective: Observe the effects of Fufangkushen injection alone and the effects when it is combined with oxaliplatin and human recombinant endostatin (endostar) on human hepatoma cell lines SMMC-7721 in vitro, and investigate the mechanism, to provide the theoretical foundation for the combination of chemotherapy with traditional medicine and antiangiogenic therapy to cure hepatocarcinoma.
Methods: Use various methods to detect the growth condition of human hepatoma cell lines SMMC-7721 in vitro when they are affected by different concentration of Fufangkushen injection, oxaliplatin and endostar alone and by the combinative use of the drugs above: (1) Estimating the inhibition of proliferation by MTT-test; (2) Observing the morphologic changes by light microscope; (3) Observing the morphologic characteristics of apoptosis by fluorescence microscope; (4) Analyzing the distribution of cell cycle by flow cytometry; (5) Observing the dyed nucleolus of apoptosis cell by TUNEL method; (6) Detecting the expressions of apoptosis-associated gene proteins: Survivin, Bcl-2 and Caspase-3 by immunohistochemical technique.
Results: (1) Fufangkushen injection could inhibit the proliferation of SMMC-7721 cell lines, and the inhibition depends on the exposure time and dose; there were synergistic effects when combine Fufangkushen injection of 50μl/ml with oxaliplatin of 1μg/ml, while no obvious effects when combined with endostar; (2) Observed by light microscope, the Cells shrank and turned round, and the contacts among cells became loose; (3) Observed by fluorescence microscope, the early and late stage apoptosis took place in cells; (4) The analysis of cell cycle showed that drugs blocked cells at G_0/G_1 phase, the proportion of cells increased at G_0/G_1 phase while it decreased at S phase; (5) The TUNEL method detected that the nucleuses of apoptotic cells were dyed deep brown, the apoptosis index (AI) increased when combined with oxaliplatin, while there was no obvious difference when combined with endostar; (6) The expressions of Survivin and Bcl-2 were up-regulated, while the expressions of Caspase-3 were down-regulated; The expressions of Survivin and Bcl-2 was increased while Caspase-3 was decreased when combined with oxaliplatin, no obvious difference was observed when combined with endostar.
Conclusion: Fufangkushen injection could inhibit the proliferation of SMMC-7721 cell lines, the effects were achieved by apoptosis related to the blocking of cell cycle at G_0/G_1 phase, down-regulation of the expressions of Caspase-3 and up-regulation of the expressions of Survivin and Bcl-2. When combined with oxaliplatin, it strengthened the inhibition of proliferation and inducement of apoptosis, while no obvious strengthening effects were observed when combined with endastar. This research provides the experimental foundation for the combination of TCM with western medicine to cure hepatocarcinoma.
方法:将不同浓度的复方苦参注射液、奥沙利铂、恩度单药以及联合作用于体外培养的人肝癌细胞株SMMC-7721,使用不同的技术检测细胞生长状况:(1)MTT法分析不同浓度和作用时间的药物对细胞增殖的抑制效果;(2)倒置显微镜观察细胞的形态学变化;(3)荧光显微镜观察细胞凋亡的形态变化特征;(4)流式细胞术检测细胞周期的分布改变;(5)原位末端标记法(TUNEL)检测凋亡细胞的细胞核着色情况;(6)免疫细胞化学法检测细胞凋亡相关蛋白Survivin、Bcl-2、Caspase-3的表达。
结果:(1)复方苦参注射液对肝癌SMMC-7721细胞有抑制增殖的作用,且该作用具有时间、剂量依赖性;50μl/ml的复方苦参注射液与1μg/ml的奥沙利铂联合作用48h后表现出协同作用,但与恩度联合未见明显增强;(2)光镜下观察,药物作用后细胞体积变小,逐渐变圆,细胞间接触变松;(3)荧光显微镜下观察,细胞发生了明显的早期凋亡和晚期凋亡,联合奥沙利铂组凋亡细胞比例显著升高;(4)流式细胞术检测显示药物组G_0/G_1期细胞比例升高,S期比例下降,细胞被阻滞于G_0/G_1期;(5)原位末端标记法(TUNEL)检测到药物作用后,凋亡细胞的细胞核被染成深褐色,当联合奥沙利铂后凋亡指数显著升高,而联合恩度未见明显变化;(6)免疫细胞化学法显示药物作用后,SMMC-7721细胞Survivin、Bcl-2蛋白的表达较对照组减弱(P＜0.01),Caspase-3的表达增高(P＜0.01),当联合奥沙利铂后,这种作用得到显著加强(P＜0.01),而联合恩度未见明显变化。
结论:复方苦参注射液对肝癌SMMC-7721细胞有抑制增殖的作用,该作用可能是通过将细胞阻滞于G_0/G_1期,同时下调Survivin、Bcl-2的表达,上调Caspase-3的表达而诱导细胞凋亡来实现的。当复方苦参注射液与奥沙利铂联合后,其抑制增殖和诱导凋亡的作用显著增强,表现出协同作用;而当联合恩度时,其作用未见明显增强。本研究为临床探索优化的中西医结合治疗原发性肝癌的联合方案提供了实验依据。
Objective: Observe the effects of Fufangkushen injection alone and the effects when it is combined with oxaliplatin and human recombinant endostatin (endostar) on human hepatoma cell lines SMMC-7721 in vitro, and investigate the mechanism, to provide the theoretical foundation for the combination of chemotherapy with traditional medicine and antiangiogenic therapy to cure hepatocarcinoma.
Methods: Use various methods to detect the growth condition of human hepatoma cell lines SMMC-7721 in vitro when they are affected by different concentration of Fufangkushen injection, oxaliplatin and endostar alone and by the combinative use of the drugs above: (1) Estimating the inhibition of proliferation by MTT-test; (2) Observing the morphologic changes by light microscope; (3) Observing the morphologic characteristics of apoptosis by fluorescence microscope; (4) Analyzing the distribution of cell cycle by flow cytometry; (5) Observing the dyed nucleolus of apoptosis cell by TUNEL method; (6) Detecting the expressions of apoptosis-associated gene proteins: Survivin, Bcl-2 and Caspase-3 by immunohistochemical technique.
Results: (1) Fufangkushen injection could inhibit the proliferation of SMMC-7721 cell lines, and the inhibition depends on the exposure time and dose; there were synergistic effects when combine Fufangkushen injection of 50μl/ml with oxaliplatin of 1μg/ml, while no obvious effects when combined with endostar; (2) Observed by light microscope, the Cells shrank and turned round, and the contacts among cells became loose; (3) Observed by fluorescence microscope, the early and late stage apoptosis took place in cells; (4) The analysis of cell cycle showed that drugs blocked cells at G_0/G_1 phase, the proportion of cells increased at G_0/G_1 phase while it decreased at S phase; (5) The TUNEL method detected that the nucleuses of apoptotic cells were dyed deep brown, the apoptosis index (AI) increased when combined with oxaliplatin, while there was no obvious difference when combined with endostar; (6) The expressions of Survivin and Bcl-2 were up-regulated, while the expressions of Caspase-3 were down-regulated; The expressions of Survivin and Bcl-2 was increased while Caspase-3 was decreased when combined with oxaliplatin, no obvious difference was observed when combined with endostar.
Conclusion: Fufangkushen injection could inhibit the proliferation of SMMC-7721 cell lines, the effects were achieved by apoptosis related to the blocking of cell cycle at G_0/G_1 phase, down-regulation of the expressions of Caspase-3 and up-regulation of the expressions of Survivin and Bcl-2. When combined with oxaliplatin, it strengthened the inhibition of proliferation and inducement of apoptosis, while no obvious strengthening effects were observed when combined with endastar. This research provides the experimental foundation for the combination of TCM with western medicine to cure hepatocarcinoma.
引文
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