本文選題：順鉑 + 細(xì)胞凋亡��； 參考：《天津醫(yī)科大學(xué)》2017年碩士論文

【摘要】：MTOR是一種細(xì)胞增殖、凋亡及生存的主要調(diào)節(jié)分子。MTOR抑制劑,如雷帕霉素,具有免疫抑制作用及抗腫瘤效應(yīng)。而依維莫司作為雷帕霉素的一種衍生物,也有著抑制MTOR活性的藥物學(xué)功能。然而,依維莫司是否有抗腫瘤效應(yīng)卻不明確。我們發(fā)現(xiàn)使用依維莫司處理人皮膚鱗狀細(xì)胞癌系COLO-16細(xì)胞后,MTOR磷酸化水平下降,LC3-I向LC3-II的轉(zhuǎn)化增加,但自噬流卻并未增強;凋亡標(biāo)志分子caspase3和PARP也并未發(fā)生剪切分裂。同時我們也發(fā)現(xiàn)依維莫司單獨處理細(xì)胞時,細(xì)胞死亡的一些關(guān)鍵通路并未受到影響;但是與順鉑共同處理細(xì)胞時,增強了順鉑誘導(dǎo)的細(xì)胞死亡。依維莫司并沒有影響順鉑誘導(dǎo)的細(xì)胞凋亡,而Chk1(檢測點激酶1)的激活卻受到了抑制。我們研究證實,依維莫司通過抑制檢測點激酶1的激活增強順鉑誘導(dǎo)COLO-16細(xì)胞死亡,而且這種效應(yīng)不依賴于凋亡和自噬調(diào)控,發(fā)現(xiàn)了依維莫司新的藥物效應(yīng)作用機制�！灸康摹刻接懸谰S莫司對順鉑的抗人皮膚鱗狀細(xì)胞癌COLO-16細(xì)胞效應(yīng)的增效機制�！痉椒ā糠謩e用50、100、200nmol/L的依維莫司和25μmol/L順鉑處理人皮膚鱗狀細(xì)胞癌系COLO-16細(xì)胞12、24小時,用AO標(biāo)記自噬體囊泡并結(jié)合溶酶體酶抑制劑分析自噬和自噬流水平。Western印跡分析自噬標(biāo)記性分子LC3-Ⅰ→LC3-Ⅱ轉(zhuǎn)化、凋亡和細(xì)胞周期相關(guān)信號通路,并檢測MTOR通路、Chk1磷酸化水平、caspase3和PARP剪切;LDH釋放實驗分析細(xì)胞死亡;AnnexinV-EGFP染色分析細(xì)胞凋亡。依維莫司與順鉑聯(lián)合處理的機制實驗通過Western印跡進(jìn)行MTOR、Akt、DNA損傷相關(guān)信號以及Chk通路分析。【結(jié)果】50、100、200nmol/L的依維莫司處理COLO-16細(xì)胞12、24小時后,MTOR2448位點和2481位點的磷酸化水平降低,Rictor1135位點磷酸化水平也降低。然而,下游信號ULK1蛋白的ser757位點的磷酸化水平、P70S6激酶的thr389位點的磷酸化水平的變化并不顯著。12小時LC3-Ⅰ→LC3-Ⅱ轉(zhuǎn)化率分別為3.52±0.21、4.03±0.39、5.05±0.22,兩兩之間比較,差異無統(tǒng)計學(xué)意義(P0.05),與未用藥物處理的對照組(2.07±0.05)比較,差異有統(tǒng)計學(xué)意義(P0.05)。24小時LC3-Ⅰ→LC3-Ⅱ轉(zhuǎn)化率分別為3.38±0.26、3.29±0.06、6.57±0.16,兩兩之間比較,差異無統(tǒng)計學(xué)意義(P0.05),與未用藥物處理的對照組(2.61±0.16)比較,差異有統(tǒng)計學(xué)意義(P0.05)。50、100、200nmol/L的依維莫司聯(lián)合E64d、pepstatin處理COLO-16細(xì)胞12小時后,LC3-Ⅱ與β肌動蛋白的比值分別為1.26±0.40、1.16±0.34、1.21±0.39,與E64d、pepstatin單獨處理細(xì)胞組(1.19±0.27)比較,差異無統(tǒng)計學(xué)意義(P0.05)。100nmol/L依維莫司聯(lián)合E64d、pepstatin自噬體囊泡表達(dá)陽性率2.06±0.61,與E64d、pepstatin單獨處理細(xì)胞組(1.68±0.62)比較,差異無統(tǒng)計學(xué)意義(P0.05)。依維莫司處理對Akt的總蛋白水平和磷酸化水平無明顯影響。順鉑單獨處理COLO-16細(xì)胞12、24小時,其中12小時細(xì)胞死亡率(%)14.33±3.07,24小時細(xì)胞死亡率(%)18.20±1.46。依維莫司單獨處理細(xì)胞12、24小時時:12小時細(xì)胞死亡率(%)0.82±0.47,24小時細(xì)胞死亡率(%)8.75±1.17,而順鉑和依維莫司聯(lián)合處理細(xì)胞12、24小時,12小時細(xì)胞死亡率(%)28.27±2.12,24小時細(xì)胞死亡率(%)42.58±0.93,細(xì)胞死亡率明顯高于順鉑單獨處理時的死亡率。順鉑處理細(xì)胞24小時后誘導(dǎo)了caspase3和PARP的剪切分裂,另外,順鉑處理細(xì)胞后,膜聯(lián)蛋白(annexin)和碘化丙啶(PI)標(biāo)記的細(xì)胞數(shù)目增多。而且,順鉑單獨處理細(xì)胞和聯(lián)合依維莫司處理細(xì)胞24小時,caspase3和PARP的剪切分裂沒有明顯的差異,膜聯(lián)蛋白(annexin)和碘化丙啶(PI)標(biāo)記的細(xì)胞數(shù)目也沒有明顯差異(P0.05)。而且,順鉑單獨處理細(xì)胞組與聯(lián)合依維莫司處理細(xì)胞組處理細(xì)胞12、24小時LC3-Ⅱ形成、caspase3和PARP剪切、AnnexinV標(biāo)記細(xì)胞數(shù)均無統(tǒng)計學(xué)差異(P0.05)。順鉑處理COLO-16細(xì)胞12、24小時后,Rictor的thr1135位點磷酸化水平和Chk1的ser345位點磷酸化水平明顯升高,依維莫司單獨處理無類似效應(yīng)。依維莫司與順鉑聯(lián)合處理后12和24小時,相對于順鉑單獨處理的細(xì)胞,上調(diào)的Chk1和Rictor磷酸化水平受到明顯抑制�！窘Y(jié)論】1.依維莫司增強順鉑誘導(dǎo)COLO-16細(xì)胞死亡,這種效應(yīng)不依賴于凋亡和自噬調(diào)控。2.COLO-16細(xì)胞MTOR通路對依維莫司處理敏感,但其下游信號并非同步產(chǎn)生級聯(lián)反應(yīng)。細(xì)胞凋亡、Akt通路對依維莫司處理不敏感。3.依維莫司通過抑制檢測點激酶1的激活增強順鉑誘導(dǎo)COLO-16細(xì)胞死亡,但無加重順鉑所導(dǎo)致的DNA損傷。
[Abstract]:MTOR is a major regulatory molecule.MTOR inhibitor for cell proliferation, apoptosis and survival, such as rapamycin, which has immunosuppressive effects and antitumor effects. As a derivative of rapamycin, iverimus also has a pharmacological function to inhibit MTOR activity. However, it is not clear whether iverimus has an antitumor effect. After the use of imimus to treat human squamous cell carcinoma COLO-16 cells, the level of phosphorylation of MTOR decreased and the transformation of LC3-I to LC3-II increased, but the autophagic flow did not increase; the apoptotic markers, Caspase3 and PARP, did not occur in shear division. The pathway was not affected, but the cell death induced by cisplatin was enhanced when cisplatin was treated together with cisplatin. Imus did not affect cisplatin induced apoptosis, but the activation of Chk1 (detection point kinase 1) was inhibited. Our study confirmed that the activation of the Imus through inhibitory detection point kinase 1 enhanced cisplatin induced COLO-1 6 cell death, and this effect is not dependent on apoptosis and autophagy regulation, and a new mechanism of effector effect of Imus is found. [Objective] to explore the synergistic mechanism of imimols on cisplatin against human skin squamous cell carcinoma COLO-16 cell effect. [Methods] the treatment was treated with 50100200nmol/L's imimus and 25 mol/L cisplatin respectively. Human skin squamous cell carcinoma cell line COLO-16 cells are 12,24 hours, using AO to mark autophagosome vesicles and combine with lysosomal enzyme inhibitors to analyze autophagic and autophagic flow level.Western blot analysis of autophagic markers LC3- I to LC3- II transformation, apoptosis and cell cycle related signal pathways, and detect MTOR pathway, Chk1 phosphorylation level, Caspase3 and PARP. Shear; LDH release assay was used to analyze cell death; AnnexinV-EGFP staining was used to analyze cell apoptosis. The mechanism of combined treatment with imimus and cisplatin was carried out by Western blot for MTOR, Akt, DNA damage related signals and Chk pathway analysis. [results] 50100200nmol/L's everimus treated COLO-16 cells for 12,24 hours, MTOR2448 site and 2 The phosphorylation level of the 481 loci was reduced and the phosphorylation level of the Rictor1135 site decreased. However, the level of phosphorylation of the ser757 loci of the downstream signal ULK1 protein, the change in the phosphorylation level of the thr389 site of the P70S6 kinase was not significantly.12 hours LC3- I to LC3- II conversion rate was 3.52 + 0.21,4.03 + 0.39,5.05 + 0.22, respectively, 22, respectively. The difference was not statistically significant (P0.05), compared with the control group (2.07 + 0.05) without drug treatment (2.07 + 0.05), the difference was statistically significant (P0.05).24 hours LC3- I to LC3- II conversion rate was 3.38 + 0.26,3.29 + 0.16 respectively, the difference was not statistically significant (P0.05), compared with the control group (2.61 + 0.16), which was not treated with drug treatment (2.61 + 0.16). The ratio of LC3- II to COLO-16 cells was 1.26 + 0.40,1.16 + 0.34,1.21 + 0.39 respectively after 12 hours of COLO-16 cells treated with.50100200nmol/L and E64d, and LC3- II and beta actin were respectively compared with E64d and pepstatin alone (1.19 + 0.27). There was no statistical difference (P0.05) with.100nmol/L imimols. E64d, pepstatin autophagic vesicle positive rate was 2.06 + 0.61, compared with E64d, pepstatin alone treated cell group (1.68 + 0.62), the difference was not statistically significant (P0.05). The total protein level and phosphorylation level of Akt were not significantly affected by the imimolus treatment. The 12 hour cell death rate of 12 hours (%) was 1 of cisplatin alone at 12,24 hours. 4.33 + 3.07,24 hours cell death rate (%) 18.20 + 1.46. imimus treated cells for 12,24 hours alone: 12 hours cell mortality (%) 0.82 + 0.47,24 hours cell mortality (%) 8.75 + 1.17, while cisplatin and everimus combined with 12,24 hours, 12 hours cell mortality (%) 28.27 + 2.12,24 hours cell mortality (%) 42.58 + 0.93, The cell death rate was significantly higher than that of cisplatin alone. 24 hours after cisplatin treated cells, the shear division of Caspase3 and PARP was induced. In addition, after cisplatin treated cells, the number of cells marked by annexin and PI was increased. Moreover, cisplatin treated cells alone and combined with ivimols for 24 hours. There was no significant difference in the shear division between Caspase3 and PARP, and there was no significant difference in the number of cells labeled by annexin (annexin) and propidium iodide (PI). Moreover, the cells treated by cisplatin alone and in the combined Imus treated cell group were treated with 12,24 hours LC3- II formation, Caspase3 and PARP shear, and AnnexinV labeled cells. No statistical difference (P0.05). After COLO-16 cells were treated with cisplatin for 12,24 hours, the level of phosphorylation of Rictor at thr1135 site and the phosphorylation level of ser345 loci of Chk1 were significantly increased. There was no similar effect by imimus alone. 12 and 24 hours after the combination of imimus and cisplatin, the up regulation of Chk1 and Ric relative to the cells treated by cisplatin alone The level of tor phosphorylation was significantly inhibited. [Conclusion] 1. imimoll enhanced cisplatin induced COLO-16 cell death, which is not dependent on apoptosis and autophagy regulating the sensitivity of the MTOR pathway to the imimus treatment, but the downstream signal is not synchronized with the cascade reaction. Cell apoptosis, Akt pathway is not sensitive to imimus treatment. Sensory.3. everolimus enhanced cisplatin induced COLO-16 cell death by inhibiting the activation of checkpoint kinase 1, but did not aggravate cisplatin induced DNA damage.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R739.5

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 陳蘭;榮冬蕓;吳春維;曹煜;;依維莫司和AR-A014418聯(lián)合使用促進(jìn)黑素瘤細(xì)胞A375的凋亡[J];中華皮膚科雜志;2016年04期

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本文編號：1852154