蒿甲醚對熱誘導口腔舌鱗癌Cal 27細胞凋亡的增敏作用及機制研究
發(fā)布時間：2018-09-17 12:00
【摘要】：[目的]探討蒿甲醚對熱誘導口腔舌鱗癌Cal27細胞凋亡的增敏作用及相關機制。 [方法](1)實驗分組：對照組(常規(guī)培養(yǎng)Cal27細胞),熱誘導組(43℃60min、43℃80min、43℃120min熱誘導處理Cal27細胞；41℃、43℃、45℃80min熱誘導處理Cal27細胞),藥物組(150mmol/L、250mmol/L、350mmol/L、450mmol/L、550mmoI/L蒿甲醚處理Cal27細胞),藥熱聯(lián)合組(150mmol/L、250mmol/L、350mmol/L、450mmol/L、550mmol/L蒿甲醚預處理30min+43℃80min熱誘導處理Cal27細胞。(2)倒置顯微鏡觀察細胞的形態(tài)學變化。(3)MTT法檢測細胞的增殖抑制率。(4)TUNEL標記流式細胞儀檢測細胞凋亡率。(5)JC-1染色流式細胞儀檢測線粒體膜電位。(6)分光光度法檢測細胞caspase-3活性。(7)蛋白芯片雜交法檢測35種細胞凋亡相關蛋白或磷酸化蛋白表達量。(8)免疫印跡檢測驗證相關蛋白的表達變化。(9)SPSS17.0統(tǒng)計軟件對數(shù)據(jù)進行統(tǒng)計學分析。 [結果](1)倒置顯微鏡下觀察發(fā)現(xiàn),單獨熱誘導或蒿甲醚處理后,Cal27細胞生長均受抑制,部分細胞皺縮、變圓,呈浮起狀；藥熱聯(lián)合處理后,細胞生長明顯受到抑制,大量細胞皺縮、變圓,呈浮起狀。(2)單獨熱誘導處理時,Cal27細胞增殖抑制率隨熱誘導溫度升高及時間延長而升高；蒿甲醚聯(lián)合熱誘導處理與單獨熱誘導或單獨蒿甲醚組處理相比,Cal27細胞增殖抑制率明顯升高,并隨蒿甲醚濃度的升高而升高(P0.05)。(3)TUNEL標記流式細胞儀檢測Cal27細胞凋亡率,結果發(fā)現(xiàn)對照組細胞凋亡率為(1.37±0.50)%,熱誘導組(43℃80min)24h后細胞凋亡率為(26.60±4.09)%,蒿甲醚組(350mmol/L蒿甲醚)24h后細胞凋亡率為(20.17±1.67)%,而藥熱聯(lián)合組,細胞凋亡率提高為(46.80±1.04)%,與單獨熱誘導或蒿甲醚組處理組相比,統(tǒng)計學上有明顯差異(P0.05)。(4)線粒體膜電位檢測發(fā)現(xiàn),熱誘導組低電位Cal27細胞占(47.1±4.3)%,蒿甲醚組低電位Cal27細胞占(27.1±3.4)%,藥熱聯(lián)合組低電位Cal27細胞占(68.0±2.7)%(p0.05)。(5)對照組Cal27細胞Caspase-3活性OD值為(0.059±0.017),熱誘導組為(0.466±0.138),蒿甲醚組為(0.258±0.122),藥熱聯(lián)合組為(0.589±0.145)(p0.05)。(6)凋亡蛋白芯片檢測發(fā)現(xiàn)：熱誘導43℃80min)處理組中Bad、Bax、Cleaved Caspase-3、Catalase、Cytochrome c、FADD、HSP60、HSP70、Phospho-p53(S392)蛋白或磷酸化蛋白表達量與對照組相比較有明顯升高(P0.05),35種凋亡相關蛋白中未發(fā)現(xiàn)表達量明顯降低的蛋白；蒿甲醚(350mmol/L)處理組中Cleaved Caspase-3、Catalase、 P21/CIP1/CDKN1A、P27/Kip1、Phospho-p53(S392)、Phospho-Radl7(S635)蛋白或磷酸化蛋白表達量與對照組相比有明顯升高(P0.05),而Pro-Caspase-3、 Claspin、Cytochrome c、FADD、HSP60、HSP70、HTRA2/Omi、SMAC/Diablo、 XIAP蛋白表達量則降低(P0.05)；蒿甲醚聯(lián)合熱誘導處理組中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達量較對照組或熱誘導組明顯升高(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達量與對照組或熱誘導組相比有明顯降低(P0.05)。免疫印跡檢測HSP60表達量變化,與芯片結果一致。 [結論]蒿甲醚對熱誘導口腔舌鱗癌Cal27細胞凋亡的具有增敏作用,與其促進熱誘導促凋亡蛋白的表達及抑制熱誘導熱休克蛋白的表達有關。
[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.
【學位授予單位】：昆明醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R739.8
本文編號：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.
【學位授予單位】：昆明醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R739.8
【參考文獻】
相關期刊論文 前10條
1 房文錚;陳錦華;歐陽學農(nóng);;香菇多糖腹腔灌注聯(lián)合熱化療治療進展期胃癌[J];東南國防醫(yī)藥;2010年02期
2 潘麓羽;曹建平;陳光烈;紀蓉;封陽;周媛媛;彭曉梅;崔丹;朱巍;樊賽軍;;青蒿素與蒿甲醚對人鼻咽癌CNE-1細胞放射增敏作用的比較研究[J];輻射研究與輻射工藝學報;2010年03期
3 張俐娜,張平義,李翔,潘蘇,朱榮平;香菇多糖的成分及其分子量研究[J];高等學�；瘜W學報;1998年09期
4 方希敏,陳銘珍,陳日玲;細胞色素C與細胞凋亡[J];國外醫(yī)學.臨床生物化學與檢驗學分冊;2005年01期
5 邵義如;朱尤慶;劉敏;;二氫青蒿素對結腸癌細胞系SW480增殖凋亡的影響[J];武漢大學學報(醫(yī)學版);2008年03期
6 孔天翰,董偉華,周永安,韓雪飛,陳華艷,吳逸明;3種蝎毒抗癌多肽制劑抑制小鼠肝癌及增強免疫的作用比較[J];河南醫(yī)科大學學報;1999年01期
7 高緒照;張長習;劉吉佳;;加溫聯(lián)合冬凌草甲素抑制肝癌細胞SMMC-7721的生長及其機制探討[J];臨床腫瘤學雜志;2007年05期
8 賀會江,祁超,黃靜,吳細丕,黃傳繼,高春芳;蝎毒的熱增敏研究[J];齊魯腫瘤雜志;1999年01期
9 謝茹燕;喬敏敏;章永平;胡梅潔;;蒿甲醚對胃癌細胞和胰腺癌細胞的體外殺傷作用[J];上海交通大學學報(醫(yī)學版);2008年03期
10 曹智;馬駿;袁文俊;林麗;;熱休克蛋白60與細胞凋亡[J];生理科學進展;2008年03期
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