蒿甲醚对热诱导口腔舌鳞癌Cal 27细胞凋亡的增敏作用及机制研究
发布时间：2018-09-17 12:00
【摘要】：[目的]探讨蒿甲醚对热诱导口腔舌鳞癌Cal27细胞凋亡的增敏作用及相关机制。 [方法](1)实验分组：对照组(常规培养Cal27细胞),热诱导组(43℃60min、43℃80min、43℃120min热诱导处理Cal27细胞；41℃、43℃、45℃80min热诱导处理Cal27细胞),药物组(150mmol/L、250mmol/L、350mmol/L、450mmol/L、550mmoI/L蒿甲醚处理Cal27细胞),药热联合组(150mmol/L、250mmol/L、350mmol/L、450mmol/L、550mmol/L蒿甲醚预处理30min+43℃80min热诱导处理Cal27细胞。(2)倒置显微镜观察细胞的形态学变化。(3)MTT法检测细胞的增殖抑制率。(4)TUNEL标记流式细胞仪检测细胞凋亡率。(5)JC-1染色流式细胞仪检测线粒体膜电位。(6)分光光度法检测细胞caspase-3活性。(7)蛋白芯片杂交法检测35种细胞凋亡相关蛋白或磷酸化蛋白表达量。(8)免疫印迹检测验证相关蛋白的表达变化。(9)SPSS17.0统计软件对数据进行统计学分析。 [结果](1)倒置显微镜下观察发现,单独热诱导或蒿甲醚处理后,Cal27细胞生长均受抑制,部分细胞皱缩、变圆,呈浮起状；药热联合处理后,细胞生长明显受到抑制,大量细胞皱缩、变圆,呈浮起状。(2)单独热诱导处理时,Cal27细胞增殖抑制率随热诱导温度升高及时间延长而升高；蒿甲醚联合热诱导处理与单独热诱导或单独蒿甲醚组处理相比,Cal27细胞增殖抑制率明显升高,并随蒿甲醚浓度的升高而升高(P0.05)。(3)TUNEL标记流式细胞仪检测Cal27细胞凋亡率,结果发现对照组细胞凋亡率为(1.37±0.50)%,热诱导组(43℃80min)24h后细胞凋亡率为(26.60±4.09)%,蒿甲醚组(350mmol/L蒿甲醚)24h后细胞凋亡率为(20.17±1.67)%,而药热联合组,细胞凋亡率提高为(46.80±1.04)%,与单独热诱导或蒿甲醚组处理组相比,统计学上有明显差异(P0.05)。(4)线粒体膜电位检测发现,热诱导组低电位Cal27细胞占(47.1±4.3)%,蒿甲醚组低电位Cal27细胞占(27.1±3.4)%,药热联合组低电位Cal27细胞占(68.0±2.7)%(p0.05)。(5)对照组Cal27细胞Caspase-3活性OD值为(0.059±0.017),热诱导组为(0.466±0.138),蒿甲醚组为(0.258±0.122),药热联合组为(0.589±0.145)(p0.05)。(6)凋亡蛋白芯片检测发现：热诱导43℃80min)处理组中Bad、Bax、Cleaved Caspase-3、Catalase、Cytochrome c、FADD、HSP60、HSP70、Phospho-p53(S392)蛋白或磷酸化蛋白表达量与对照组相比较有明显升高(P0.05),35种凋亡相关蛋白中未发现表达量明显降低的蛋白；蒿甲醚(350mmol/L)处理组中Cleaved Caspase-3、Catalase、 P21/CIP1/CDKN1A、P27/Kip1、Phospho-p53(S392)、Phospho-Radl7(S635)蛋白或磷酸化蛋白表达量与对照组相比有明显升高(P0.05),而Pro-Caspase-3、 Claspin、Cytochrome c、FADD、HSP60、HSP70、HTRA2/Omi、SMAC/Diablo、 XIAP蛋白表达量则降低(P0.05)；蒿甲醚联合热诱导处理组中Bad、Bax、Cleaved Caspase-3、Catalase、Fas/TNFRSF6/CD、P21/CIP1/CDKN1A、P27/Kip1、 Phospho-p53(S15)、Phospho-p53(S46)、Phospho-p53(S392)、Phospho-Radl7(S635)蛋白或磷酸化蛋白表达量较对照组或热诱导组明显升高(P0.05),而Pro-Caspase-3、Cytochrome c、FADD、HSP60、HSP70、HTRA2/Omi、SMAC/Diablo、 XIAP蛋白或磷酸化蛋白表达量与对照组或热诱导组相比有明显降低(P0.05)。免疫印迹检测HSP60表达量变化,与芯片结果一致。 [结论]蒿甲醚对热诱导口腔舌鳞癌Cal27细胞凋亡的具有增敏作用,与其促进热诱导促凋亡蛋白的表达及抑制热诱导热休克蛋白的表达有关。
[Abstract]:[Objective] to explore the sensitizing effect of artemether on heat induced apoptosis of oral tongue squamous cell carcinoma Cal27 cells and its related mechanisms.
[Methods] (1) The experimental group was divided into control group (Cal27 cells were cultured routinely), heat-induced group (Cal27 cells were treated at 43 60 min, 43 80 min, 43 120 min; Cal27 cells were treated at 41 43 80 min, 45 80 min), drug group (150 mmol / L, 250 mmol / L, 350 mmol / L, 450 mmol / L, 550 mmoI / L artemether treated Cal27 cells), drug-heat combined group (150 mmol / L, 45 mmol / L, 80 min). Cal27 cells were pretreated with 250 mmol/L, 350 mmol/L, 450 mmol/L, 550 mmol/L artemether for 30 min + 43 65507 The activity of Caspase-3 was detected by spectrophotometry. (7) The expression of 35 apoptosis-related proteins or phosphorylated proteins was detected by protein chip hybridization. (8) The expression of related proteins was verified by Western blotting. (9) SPSS17.0 statistical software was used to analyze the data.
[Results] (1) Under inverted microscope, the growth of Cal27 cells was inhibited by heat-induced or artemether-treated alone, and some cells were shrunk, rounded and floated. After heat-treated, the growth of Cal27 cells was obviously inhibited, and a large number of cells were shrunk, rounded and floated. (2) When heat-induced alone, the inhibition rate of Cal27 cell proliferation was increased with the increase of heat-treated cells. The inhibition rate of Cal27 cell proliferation increased significantly with the increase of artemether concentration (P 0.05). (3) The apoptosis rate of Cal27 cells in control group was detected by TUNEL labeled flow cytometry. The apoptosis rate was (1.37 65 (4) Mitochondrial membrane potential test showed that low-potential Cal27 cells accounted for (47.1 6550 (6) The expression of Bad, Bax, Cleaved Caspase-3, Catalase, Cytochrome c, FADD, HSP60, HSP70, Phospho-p53 (S392) protein or phosphorylated protein was significantly higher in the heat-induced group than in the control group (P 0.05). No significant decrease was found in the protein expression; the protein expression levels of Cleaved Caspase-3, Catalase, P21/CIP1/CDKN1A, P27/Kip1, Phospho-p53 (S392), Phospho-Radl7 (S635) or phosphorylated protein in artemether (350 mmol/L) treatment group were significantly higher than those in control group (P 0.05), while the protein expression levels of Pro-Caspase-3, Claspin, Cytochrome c, FADD, HSP60, HSP60, and HSP60 were significantly higher than those in control group (P 0.05). The expression of Bad, Bax, Cleaved Caspase-3, Catalase, Fas/TNFRSF6/CD, P21/CIP1/CDKN1A, P27/Kip1, Phospho-p53 (S15), Phospho-p53 (S46), Phospho-p53 (S392), Phospho-Radl7 (S635) protein or phosphorylated protein in artemether combined with heat-induced group were lower than those in control group or heat-induced group. The expression of Pro-Caspase-3, Cytochrome c, FADD, HSP60, HSP70, HTRA2/Omi, SMAC/Diablo, XIAP protein or phosphorylation protein in the induction group was significantly higher than that in the control group or the heat-induced group (P 0.05). The expression of HSP60 was detected by Western blot, which was consistent with the results of the chip.
[Conclusion] Artemether has a sensitizing effect on heat-induced apoptosis of oral tongue squamous cell carcinoma Cal27 cells, which is related to its promotion of heat-induced pro-apoptotic protein expression and inhibition of heat-induced heat shock protein expression.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.8
本文编号：2245847
[Abstract]:[Objective] to explore the sensitizing effect of artemether on heat induced apoptosis of oral tongue squamous cell carcinoma Cal27 cells and its related mechanisms.
[Methods] (1) The experimental group was divided into control group (Cal27 cells were cultured routinely), heat-induced group (Cal27 cells were treated at 43 60 min, 43 80 min, 43 120 min; Cal27 cells were treated at 41 43 80 min, 45 80 min), drug group (150 mmol / L, 250 mmol / L, 350 mmol / L, 450 mmol / L, 550 mmoI / L artemether treated Cal27 cells), drug-heat combined group (150 mmol / L, 45 mmol / L, 80 min). Cal27 cells were pretreated with 250 mmol/L, 350 mmol/L, 450 mmol/L, 550 mmol/L artemether for 30 min + 43 65507 The activity of Caspase-3 was detected by spectrophotometry. (7) The expression of 35 apoptosis-related proteins or phosphorylated proteins was detected by protein chip hybridization. (8) The expression of related proteins was verified by Western blotting. (9) SPSS17.0 statistical software was used to analyze the data.
[Results] (1) Under inverted microscope, the growth of Cal27 cells was inhibited by heat-induced or artemether-treated alone, and some cells were shrunk, rounded and floated. After heat-treated, the growth of Cal27 cells was obviously inhibited, and a large number of cells were shrunk, rounded and floated. (2) When heat-induced alone, the inhibition rate of Cal27 cell proliferation was increased with the increase of heat-treated cells. The inhibition rate of Cal27 cell proliferation increased significantly with the increase of artemether concentration (P 0.05). (3) The apoptosis rate of Cal27 cells in control group was detected by TUNEL labeled flow cytometry. The apoptosis rate was (1.37 65 (4) Mitochondrial membrane potential test showed that low-potential Cal27 cells accounted for (47.1 6550 (6) The expression of Bad, Bax, Cleaved Caspase-3, Catalase, Cytochrome c, FADD, HSP60, HSP70, Phospho-p53 (S392) protein or phosphorylated protein was significantly higher in the heat-induced group than in the control group (P 0.05). No significant decrease was found in the protein expression; the protein expression levels of Cleaved Caspase-3, Catalase, P21/CIP1/CDKN1A, P27/Kip1, Phospho-p53 (S392), Phospho-Radl7 (S635) or phosphorylated protein in artemether (350 mmol/L) treatment group were significantly higher than those in control group (P 0.05), while the protein expression levels of Pro-Caspase-3, Claspin, Cytochrome c, FADD, HSP60, HSP60, and HSP60 were significantly higher than those in control group (P 0.05). The expression of Bad, Bax, Cleaved Caspase-3, Catalase, Fas/TNFRSF6/CD, P21/CIP1/CDKN1A, P27/Kip1, Phospho-p53 (S15), Phospho-p53 (S46), Phospho-p53 (S392), Phospho-Radl7 (S635) protein or phosphorylated protein in artemether combined with heat-induced group were lower than those in control group or heat-induced group. The expression of Pro-Caspase-3, Cytochrome c, FADD, HSP60, HSP70, HTRA2/Omi, SMAC/Diablo, XIAP protein or phosphorylation protein in the induction group was significantly higher than that in the control group or the heat-induced group (P 0.05). The expression of HSP60 was detected by Western blot, which was consistent with the results of the chip.
[Conclusion] Artemether has a sensitizing effect on heat-induced apoptosis of oral tongue squamous cell carcinoma Cal27 cells, which is related to its promotion of heat-induced pro-apoptotic protein expression and inhibition of heat-induced heat shock protein expression.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.8
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