苯吡啶缩氨基硫脲抗肿瘤作用及其机制研究
摘要
目的:探讨苯吡啶缩氨基硫脲对人白血病K562细胞的增殖抑制作用及其可能机制。
方法:以新型化合物苯吡啶缩氨基硫脲为研究对象,以顺铂为阳性对照药物,通过MTT比色法、LDH漏出率、台盼蓝据染法观察苯吡啶缩氨基硫脲对K562细胞株体外增殖的抑制作用,并用MTT法和LDH法观察苯吡啶缩氨基硫脲对人正常肝chang liver细胞株体外增殖的抑制作用,考察苯吡啶缩氨基硫脲的体外抗肿瘤活性及其选择性。通过AO/EB染色法利用荧光倒置显微镜观察K562细胞的凋亡形态,Rh123染色法利用荧光分光光度计检测细胞线粒体膜电位的水平变化,钙离子敏感的荧光探针Fura-2/AM利用荧光分光光度计检测K562细胞内游离Ca2+浓度的变化,Hoechst33342/PI染色法利用HCS检测K562细胞的凋亡,从细胞水平探讨苯吡啶缩氨基硫脲的诱导K562细胞凋亡的机制。
结果:通过MTT比色法,采用不同浓度的BT处理K562细胞,作用时间分别为24h、36h、48h。结果表明,BT对K562细胞的成长呈现抑制作用,且该作用表现为明显的剂量依赖性和时间依赖性;相同浓度(50μM)下,BT的作用强于CDDP。BT作用于K562细胞48h时,IC50为19.62μM。BT对张氏肝细胞的生长基本无抑制作用。BT使K562细胞的LDH漏出率增加,而对张氏肝细胞的LDH漏出率基本无影响。生长曲线表明,BT对K562细胞的生长呈现抑制作用。AO/EB染色法观察细胞的形态学表明,BT可促进K562细胞凋亡,出现典型的凋亡形态学特征。Rh123染色法测定细胞的线粒体膜电位的变化可看到BT可降低K562细胞的线粒体膜电位。Fura-2/AM结合荧光分光光度计检测不同波长下的荧光强度计算出相应细胞中的Ca2+浓度表明,在1.3mmol/L细胞外Ca2+的情况下, K562细胞正常组的[Ca2+]i为219.2nmol/L ,BT使K562细胞内[Ca2+]i升高。Hoechst33342/PI染色法测定K562细胞的凋亡实验表明,由HCS检测到的细胞的荧光强度值随着BT浓度的增加而逐渐增加,由HCS拍摄到的细胞形态学图片可以看到出现典型的凋亡形态学特征,且凋亡细胞数目显著增多。
结论:
新型化合物BT可引起人白血病K562细胞显著的损伤和凋亡,且在相同浓度下强于阳性对照药顺铂对K562细胞的生长抑制作用,同时BT对K562细胞的抑制作用呈现明显的浓度依赖性和时间依赖性。
Objective: To investigate the the protective anti-tumor effect and its Mechanisms of 2-benzoylpyridine thiosemicarbazone in k562 cells.
Methods: With new compounds 2-benzoylpyridine thiosemicarbazone as the research object, Cisplatin as a positive control drug, observe the 2-benzoylpyridine thiosemicarbazone’s proliferation effect in vitro on K562 cells through MTT assay,LDH leakage rate and the growth curve,observe the 2-benzoylpyridine thiosemicarbazone’s proliferation effect in vitro on chang liver cells through MTT assay and LDH leakage rate,and investigate the anti-tumor activity and selectivity of 2-benzoylpyridine thiosemicarbazone in vitro. Detecting the apoptotic morphology in K562 cells through AO/EB staining by fluorescence microscope, detecting the mitochondrial membrane potential in K562 cells through Rh123 staining by fluorescence spectrophotometer,detecting the free intracellular calcium concentrationa in K562 cells through Fura-2/AM by fluorescence spectrophotometer,detecting the apoptosis in K562 cells through Hoechst33342/PI staining by HCS,investigate the mechanism of inducing tumor cell apoptosis of 2-benzoylpyridine thiosemicarbazone from the cell lever.
Results: By MTT assay,after K562 cells treated with BT at different concentrations, the role of time is 24h、36h、48h, the results show that the proliferation of K562 cells are inhibited by BT,and the effect is showed the dose-dependent and time-dependent. Under the same concentration (50μM), BT is stronger than the role of CDDP. When acting on K562 cells 48h by BT, IC50 is 19.62μM. But the growth of Chang liver cells are hardly inhibited by BT. BT can cause the LDH leakage rate of K562 cells increasing,the growth of Chang liver cells almost are hardly inhibited by BT, LDH leakage rate is almost unchanged.The cell growth curve shows that the proliferation of K562 cells are inhibited by BT. From observing the morphological characteristics of K562 cells by AO/EB stainingthe the results show BT can promote apoptosis of K562 cells,cause the typical morphological features of apoptosis.Detecting the change s of MMP of K562 cells by Rh123 staining by the fluorescence spectrophotometer, the results show that BT can reduce MMP of K562 cells. Detecting fluorescence intensity under different wavelength spectrophotometer through the application of calcium-sensitive fluorescent probe Fura-2/AM by fluorescence spectrophotometer and calculating [Ca2+]I, the results show that, under the case of 1.3mmol/L extracellular Ca2+, [Ca2+]i was 219.2 nmol/L in normal K562 cells, [Ca2+]i in K562 cells increase in different degrees after K562 cells treated with BT.Determinating the apoptosis of K562 cells by Hoe-chest33342/PI staining,the results show that:the fluorescence intensities are increased following by increasing concentration of BT, the morphological pictures are showed the characteristics of apoptosis increase.
Conclusions: The new compound BT can cause significant damage and apoptosis in K562 cells, and the effect is showed the dose-dependent and time-dependent.
方法:以新型化合物苯吡啶缩氨基硫脲为研究对象,以顺铂为阳性对照药物,通过MTT比色法、LDH漏出率、台盼蓝据染法观察苯吡啶缩氨基硫脲对K562细胞株体外增殖的抑制作用,并用MTT法和LDH法观察苯吡啶缩氨基硫脲对人正常肝chang liver细胞株体外增殖的抑制作用,考察苯吡啶缩氨基硫脲的体外抗肿瘤活性及其选择性。通过AO/EB染色法利用荧光倒置显微镜观察K562细胞的凋亡形态,Rh123染色法利用荧光分光光度计检测细胞线粒体膜电位的水平变化,钙离子敏感的荧光探针Fura-2/AM利用荧光分光光度计检测K562细胞内游离Ca2+浓度的变化,Hoechst33342/PI染色法利用HCS检测K562细胞的凋亡,从细胞水平探讨苯吡啶缩氨基硫脲的诱导K562细胞凋亡的机制。
结果:通过MTT比色法,采用不同浓度的BT处理K562细胞,作用时间分别为24h、36h、48h。结果表明,BT对K562细胞的成长呈现抑制作用,且该作用表现为明显的剂量依赖性和时间依赖性;相同浓度(50μM)下,BT的作用强于CDDP。BT作用于K562细胞48h时,IC50为19.62μM。BT对张氏肝细胞的生长基本无抑制作用。BT使K562细胞的LDH漏出率增加,而对张氏肝细胞的LDH漏出率基本无影响。生长曲线表明,BT对K562细胞的生长呈现抑制作用。AO/EB染色法观察细胞的形态学表明,BT可促进K562细胞凋亡,出现典型的凋亡形态学特征。Rh123染色法测定细胞的线粒体膜电位的变化可看到BT可降低K562细胞的线粒体膜电位。Fura-2/AM结合荧光分光光度计检测不同波长下的荧光强度计算出相应细胞中的Ca2+浓度表明,在1.3mmol/L细胞外Ca2+的情况下, K562细胞正常组的[Ca2+]i为219.2nmol/L ,BT使K562细胞内[Ca2+]i升高。Hoechst33342/PI染色法测定K562细胞的凋亡实验表明,由HCS检测到的细胞的荧光强度值随着BT浓度的增加而逐渐增加,由HCS拍摄到的细胞形态学图片可以看到出现典型的凋亡形态学特征,且凋亡细胞数目显著增多。
结论:
新型化合物BT可引起人白血病K562细胞显著的损伤和凋亡,且在相同浓度下强于阳性对照药顺铂对K562细胞的生长抑制作用,同时BT对K562细胞的抑制作用呈现明显的浓度依赖性和时间依赖性。
Objective: To investigate the the protective anti-tumor effect and its Mechanisms of 2-benzoylpyridine thiosemicarbazone in k562 cells.
Methods: With new compounds 2-benzoylpyridine thiosemicarbazone as the research object, Cisplatin as a positive control drug, observe the 2-benzoylpyridine thiosemicarbazone’s proliferation effect in vitro on K562 cells through MTT assay,LDH leakage rate and the growth curve,observe the 2-benzoylpyridine thiosemicarbazone’s proliferation effect in vitro on chang liver cells through MTT assay and LDH leakage rate,and investigate the anti-tumor activity and selectivity of 2-benzoylpyridine thiosemicarbazone in vitro. Detecting the apoptotic morphology in K562 cells through AO/EB staining by fluorescence microscope, detecting the mitochondrial membrane potential in K562 cells through Rh123 staining by fluorescence spectrophotometer,detecting the free intracellular calcium concentrationa in K562 cells through Fura-2/AM by fluorescence spectrophotometer,detecting the apoptosis in K562 cells through Hoechst33342/PI staining by HCS,investigate the mechanism of inducing tumor cell apoptosis of 2-benzoylpyridine thiosemicarbazone from the cell lever.
Results: By MTT assay,after K562 cells treated with BT at different concentrations, the role of time is 24h、36h、48h, the results show that the proliferation of K562 cells are inhibited by BT,and the effect is showed the dose-dependent and time-dependent. Under the same concentration (50μM), BT is stronger than the role of CDDP. When acting on K562 cells 48h by BT, IC50 is 19.62μM. But the growth of Chang liver cells are hardly inhibited by BT. BT can cause the LDH leakage rate of K562 cells increasing,the growth of Chang liver cells almost are hardly inhibited by BT, LDH leakage rate is almost unchanged.The cell growth curve shows that the proliferation of K562 cells are inhibited by BT. From observing the morphological characteristics of K562 cells by AO/EB stainingthe the results show BT can promote apoptosis of K562 cells,cause the typical morphological features of apoptosis.Detecting the change s of MMP of K562 cells by Rh123 staining by the fluorescence spectrophotometer, the results show that BT can reduce MMP of K562 cells. Detecting fluorescence intensity under different wavelength spectrophotometer through the application of calcium-sensitive fluorescent probe Fura-2/AM by fluorescence spectrophotometer and calculating [Ca2+]I, the results show that, under the case of 1.3mmol/L extracellular Ca2+, [Ca2+]i was 219.2 nmol/L in normal K562 cells, [Ca2+]i in K562 cells increase in different degrees after K562 cells treated with BT.Determinating the apoptosis of K562 cells by Hoe-chest33342/PI staining,the results show that:the fluorescence intensities are increased following by increasing concentration of BT, the morphological pictures are showed the characteristics of apoptosis increase.
Conclusions: The new compound BT can cause significant damage and apoptosis in K562 cells, and the effect is showed the dose-dependent and time-dependent.
引文
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