【摘要】：[目的]探討蒿甲醚對(duì)熱誘導(dǎo)口腔舌鱗癌Cal27細(xì)胞凋亡的增敏作用及相關(guān)機(jī)制。 [方法](1)實(shí)驗(yàn)分組：對(duì)照組(常規(guī)培養(yǎng)Cal27細(xì)胞),熱誘導(dǎo)組(43℃60min、43℃80min、43℃120min熱誘導(dǎo)處理Cal27細(xì)胞；41℃、43℃、45℃80min熱誘導(dǎo)處理Cal27細(xì)胞),藥物組(150mmol/L、250mmol/L、350mmol/L、450mmol/L、550mmoI/L蒿甲醚處理Cal27細(xì)胞),藥熱聯(lián)合組(150mmol/L、250mmol/L、350mmol/L、450mmol/L、550mmol/L蒿甲醚預(yù)處理30min+43℃80min熱誘導(dǎo)處理Cal27細(xì)胞。(2)倒置顯微鏡觀察細(xì)胞的形態(tài)學(xué)變化。(3)MTT法檢測(cè)細(xì)胞的增殖抑制率。(4)TUNEL標(biāo)記流式細(xì)胞儀檢測(cè)細(xì)胞凋亡率。(5)JC-1染色流式細(xì)胞儀檢測(cè)線粒體膜電位。(6)分光光度法檢測(cè)細(xì)胞caspase-3活性。(7)蛋白芯片雜交法檢測(cè)35種細(xì)胞凋亡相關(guān)蛋白或磷酸化蛋白表達(dá)量。(8)免疫印跡檢測(cè)驗(yàn)證相關(guān)蛋白的表達(dá)變化。(9)SPSS17.0統(tǒng)計(jì)軟件對(duì)數(shù)據(jù)進(jìn)行統(tǒng)計(jì)學(xué)分析。 [結(jié)果](1)倒置顯微鏡下觀察發(fā)現(xiàn),單獨(dú)熱誘導(dǎo)或蒿甲醚處理后,Cal27細(xì)胞生長(zhǎng)均受抑制,部分細(xì)胞皺縮、變圓,呈浮起狀；藥熱聯(lián)合處理后,細(xì)胞生長(zhǎng)明顯受到抑制,大量細(xì)胞皺縮、變圓,呈浮起狀。(2)單獨(dú)熱誘導(dǎo)處理時(shí),Cal27細(xì)胞增殖抑制率隨熱誘導(dǎo)溫度升高及時(shí)間延長(zhǎng)而升高；蒿甲醚聯(lián)合熱誘導(dǎo)處理與單獨(dú)熱誘導(dǎo)或單獨(dú)蒿甲醚組處理相比,Cal27細(xì)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(biāo)記流式細(xì)胞儀檢測(cè)Cal27細(xì)胞凋亡率,結(jié)果發(fā)現(xiàn)對(duì)照組細(xì)胞凋亡率為(1.37±0.50)%,熱誘導(dǎo)組(43℃80min)24h后細(xì)胞凋亡率為(26.60±4.09)%,蒿甲醚組(350mmol/L蒿甲醚)24h后細(xì)胞凋亡率為(20.17±1.67)%,而藥熱聯(lián)合組,細(xì)胞凋亡率提高為(46.80±1.04)%,與單獨(dú)熱誘導(dǎo)或蒿甲醚組處理組相比,統(tǒng)計(jì)學(xué)上有明顯差異(P0.05)。(4)線粒體膜電位檢測(cè)發(fā)現(xiàn),熱誘導(dǎo)組低電位Cal27細(xì)胞占(47.1±4.3)%,蒿甲醚組低電位Cal27細(xì)胞占(27.1±3.4)%,藥熱聯(lián)合組低電位Cal27細(xì)胞占(68.0±2.7)%(p0.05)。(5)對(duì)照組Cal27細(xì)胞Caspase-3活性O(shè)D值為(0.059±0.017),熱誘導(dǎo)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(cè)發(fā)現(xiàn)：熱誘導(dǎo)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(dá)量與對(duì)照組相比較有明顯升高(P0.05),35種凋亡相關(guān)蛋白中未發(fā)現(xiàn)表達(dá)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(dá)量與對(duì)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(dá)量則降低(P0.05)；蒿甲醚聯(lián)合熱誘導(dǎo)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(dá)量較對(duì)照組或熱誘導(dǎo)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(dá)量與對(duì)照組或熱誘導(dǎo)組相比有明顯降低(P0.05)。免疫印跡檢測(cè)HSP60表達(dá)量變化,與芯片結(jié)果一致。 [結(jié)論]蒿甲醚對(duì)熱誘導(dǎo)口腔舌鱗癌Cal27細(xì)胞凋亡的具有增敏作用,與其促進(jìn)熱誘導(dǎo)促凋亡蛋白的表達(dá)及抑制熱誘導(dǎo)熱休克蛋白的表達(dá)有關(guān)。
[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(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R739.8

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