蛋白激酶B抑制劑派立福辛聯(lián)合神經(jīng)酰胺C6抗人腦膠質(zhì)瘤的機(jī)制研究
發(fā)布時(shí)間：2018-11-08 07:00
【摘要】：目的:觀察蛋白激酶B抑制劑哌立福辛(perifosine)、神經(jīng)酰胺C6(ceramide)對(duì)腦膠質(zhì)瘤細(xì)胞增殖、凋亡、自噬等生物學(xué)行為的影響,探討哌立福辛與神經(jīng)酰胺C6聯(lián)合用藥的協(xié)同機(jī)制,為腦膠質(zhì)瘤的靶向治療提供理論依據(jù)。方法:1.對(duì)膠質(zhì)瘤細(xì)胞予以不同濃度及時(shí)間的哌立福辛處理,運(yùn)用MTT法及細(xì)胞集落實(shí)驗(yàn)檢測(cè)腫瘤細(xì)胞增殖活性;組蛋白DNA ELISA法及Annexin V/PI雙染法檢測(cè)腫瘤細(xì)胞凋亡改變;Western-Blot法檢測(cè)AKT1、p-AKT、p-S6、p-AMPKα、p-ACC以及ATG-5、LC-3B、Beclin-1的表達(dá)情況;高效液相色譜法檢測(cè)膠質(zhì)瘤細(xì)胞內(nèi)神經(jīng)酰胺的水平的變化。2.對(duì)膠質(zhì)瘤細(xì)胞予以不同濃度及時(shí)間的神經(jīng)酰胺C6處理,MTT法檢測(cè)其對(duì)細(xì)胞活力的影響;神經(jīng)酰胺C6、哌立福辛以及二者聯(lián)合處理膠質(zhì)瘤細(xì)胞,運(yùn)用MTT法、Trypan blue實(shí)驗(yàn)檢測(cè)腫瘤細(xì)胞活性改變;Western-Blot法檢測(cè)ERK、p-AKT、p-S6、p-S6K、p-ERK以及ATG-5、LC-3B、Beclin-1的表達(dá)情況。3.運(yùn)用自噬抑制劑3-MA、氯喹(Cq)和caspase抑制劑z-VAD-fmk對(duì)上述各組細(xì)胞予以干預(yù),檢測(cè)腫瘤的細(xì)胞活力變化;對(duì)膠質(zhì)瘤細(xì)胞予以3-MA聯(lián)合哌立福辛處理,MTT法檢測(cè)腫瘤細(xì)胞活性,組蛋白DNA ELISA法及Annexin V/PI雙染法檢測(cè)腫瘤細(xì)胞凋亡改變;同樣方法檢測(cè)神經(jīng)酰胺C6聯(lián)合哌立福辛對(duì)膠質(zhì)瘤細(xì)胞凋亡的影響。4.構(gòu)建ATG5-si RNA轉(zhuǎn)染HEK293細(xì)胞株建立自噬缺陷細(xì)胞;MTT法檢測(cè)哌立福辛及神經(jīng)酰胺C6對(duì)自噬缺陷細(xì)胞活性影響,Annexin V/PI雙染法檢測(cè)其凋亡改變。結(jié)果:1.MTT法和細(xì)胞集落實(shí)驗(yàn)結(jié)果顯示哌立福辛顯著抑制膠質(zhì)瘤細(xì)胞增殖,其抑制效應(yīng)呈時(shí)間和濃度依賴性;Annexin V/PI雙染法及組蛋白DNA ELISA法提示哌立福辛能夠誘導(dǎo)膠質(zhì)瘤細(xì)胞凋亡,但誘導(dǎo)作用不明顯;Western-Blot法檢測(cè)到哌立福辛下調(diào)p-AKT的表達(dá),上調(diào)自噬相關(guān)蛋白ATG-5、LC-3B、Beclin-1蛋白表達(dá);高效液相色譜法結(jié)果顯示哌立福辛能夠輕度提高細(xì)胞內(nèi)神經(jīng)酰胺的水平。2.MTT法以及Trypan blue實(shí)驗(yàn)結(jié)果顯示神經(jīng)酰胺C6聯(lián)合哌立福辛對(duì)膠質(zhì)瘤細(xì)胞增殖抑制具有協(xié)同效應(yīng);組蛋白DNA ELISA法及Annexin V/PI雙染法結(jié)果表明膠質(zhì)瘤細(xì)胞凋亡誘導(dǎo)顯著增加;Western-Blot法結(jié)果表明聯(lián)合用藥抑制自噬相關(guān)蛋白ATG-5、LC-3B、Beclin-1蛋白表達(dá),對(duì)p-AKT影響不明顯。3.自噬抑制劑3-MA,Cq顯著抑制膠質(zhì)瘤細(xì)胞活性,但抑制作用可被z-VAD-fmk阻斷,3-MA可增強(qiáng)哌立福辛對(duì)膠質(zhì)瘤的細(xì)胞增殖抑制效應(yīng);組蛋白DNA ELISA法及Annexin V/PI雙染法顯示神經(jīng)酰胺C6可顯著增強(qiáng)哌立福辛對(duì)膠質(zhì)瘤細(xì)胞的凋亡誘導(dǎo),z-VAD-fmk可阻斷聯(lián)合用藥的協(xié)同作用。4.成功構(gòu)建自噬缺陷細(xì)胞;哌立福辛對(duì)自噬缺陷細(xì)胞的增殖抑制作用顯著增強(qiáng),聯(lián)合神經(jīng)酰胺C6未見(jiàn)明顯的協(xié)同效應(yīng);Annexin V/PI雙染法檢測(cè)結(jié)果顯示哌立福辛對(duì)自噬缺陷細(xì)胞凋亡誘導(dǎo)作用明顯增強(qiáng)。結(jié)論:1.哌立福辛有望作為膠質(zhì)瘤分子靶向治療治療藥物候選之一,具有潛在的臨床應(yīng)用價(jià)值。2.神經(jīng)酰胺C6增強(qiáng)哌立福辛對(duì)膠質(zhì)瘤細(xì)胞的殺傷作用,具有協(xié)同效應(yīng)。3.協(xié)同作用的機(jī)制是派立福辛抑制膠質(zhì)瘤細(xì)胞增殖的同時(shí)誘導(dǎo)自噬反應(yīng),自噬抑制細(xì)胞凋亡。而神經(jīng)酰胺C6能夠抑制自噬,恢復(fù)派立福辛導(dǎo)致的膠質(zhì)瘤細(xì)胞凋亡作用,增強(qiáng)派立福辛對(duì)膠質(zhì)瘤細(xì)胞的細(xì)胞殺傷作用而不依賴AKT-m TOR信號(hào)傳導(dǎo)通路。
[Abstract]:Objective: To observe the effects of protein kinase B (protein kinase B) inhibitor, such as perifosine and ceraimide on the proliferation, apoptosis and autophagy of human glioma cells, and to explore the cooperative mechanism of the combination of Lirifamin and neuropolyamine (C6). and provides a theoretical basis for the targeted treatment of the glioma. Method: 1. The proliferation activity of tumor cells was detected by MTT and Annexin V/ PI double-staining. The apoptosis of tumor cells was detected by the method of MTT and Annexin V/ PI. The expression of AKT1, p-AKT, p-S6, p-AMPK, p-ACC and ATG-5, LC-3B were detected by Western-Blot method. Bechlin-1 expression; high performance liquid chromatography for the detection of changes in the level of neurolepin in glioma cells. The effects of different concentration and time on the cell viability of the glioma cells were measured by MTT method. The cells of glioma were treated by MTT method, and the activity of the tumor cells was detected by the method of MTT, and the results of Western-Blot were used to detect ERK, p-AKT. Expression of p-S6, p-S6K, p-ERK, and ATG-5, LC-3B, and Beclin-1. the cell activity of the tumor is detected by using the self-autophagy inhibitor 3-MA, the chlorine peroxide (Cq) and the caspase inhibitor z-VAD-fmk to detect the change of the cell viability of the tumor, The apoptosis of the tumor cells was detected by the histone-DNA-ELISA and the Annexin V/ PI double-staining method. HK293 cell line was transfected with ATG5-si RNA to establish autophagy-deficient cells. MTT assay was used to detect the changes of cell activity of self-tophagy-deficient cells, and Annexin V/ PI double-staining method was used to detect the changes of apoptosis. Results: 1. The results of MTT and cell-colony experiments showed that the cell proliferation of glioma cells was significantly inhibited by the method of MTT and cell-colony, and the inhibitory effect was time-and concentration-dependent; Annexin V/ PI double-staining and histone-DNA-ELISA suggested that it was possible to induce the apoptosis of glioma cells, but the induction effect was not obvious; The expression of p-AKT was down-regulated by Western-Blot method, and the expression of autophagy-related protein ATG-5, LC-3B and Beclin-1 was up-regulated. The results of high performance liquid chromatography (HPLC) showed that the level of the neuroleptic amine in the cells could be slightly increased by the method of high performance liquid chromatography (HPLC). The results of ELISA and Annexin V/ PI double-staining showed that the apoptosis induction of glioma cells increased significantly. The results of Western-Blot show that the combination of the combination of the anti-autophagy-related protein ATG-5, LC-3B and Beclin-1 protein has no obvious effect on p-AKT. 3-MA and Cq inhibit the activity of glioma cells, but the inhibitory effect can be blocked by z-VAD-fmk, and 3-MA can enhance the inhibitory effect on the cell proliferation of glioblastoma. Both the histone DNA-linked immunosorbent assay (ELISA) and the Annexin V/ PI double-staining method can significantly enhance the induction of the apoptosis in the glioma cells, and the z-VAD-fmk can block the synergistic effect of the combination. The results of Annexin V/ PI double-staining showed that the induction of autophagy in the autophagy-deficient cells was significantly enhanced by the results of Annexin V/ PI double-staining. Conclusion: 1. Lirifamin is expected to be one of the candidate for the treatment of glioma, and has potential clinical application value. Neuralamine C6 enhances the killing of glioma cells and has a synergistic effect. The mechanism of the synergistic effect is to induce the autophagy reaction and the autophagy to inhibit the cell apoptosis while suppressing the proliferation of the glioma cells. and the neuropolyamine C6 is capable of inhibiting autophagy, restoring the apoptosis of the glioma cells caused by the palifosinine, and enhancing the cell killing effect of the palifosine on the glioma cells without relying on the AKT-m TOR signal conduction path.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R739.41
本文編號(hào)：2317726
[Abstract]:Objective: To observe the effects of protein kinase B (protein kinase B) inhibitor, such as perifosine and ceraimide on the proliferation, apoptosis and autophagy of human glioma cells, and to explore the cooperative mechanism of the combination of Lirifamin and neuropolyamine (C6). and provides a theoretical basis for the targeted treatment of the glioma. Method: 1. The proliferation activity of tumor cells was detected by MTT and Annexin V/ PI double-staining. The apoptosis of tumor cells was detected by the method of MTT and Annexin V/ PI. The expression of AKT1, p-AKT, p-S6, p-AMPK, p-ACC and ATG-5, LC-3B were detected by Western-Blot method. Bechlin-1 expression; high performance liquid chromatography for the detection of changes in the level of neurolepin in glioma cells. The effects of different concentration and time on the cell viability of the glioma cells were measured by MTT method. The cells of glioma were treated by MTT method, and the activity of the tumor cells was detected by the method of MTT, and the results of Western-Blot were used to detect ERK, p-AKT. Expression of p-S6, p-S6K, p-ERK, and ATG-5, LC-3B, and Beclin-1. the cell activity of the tumor is detected by using the self-autophagy inhibitor 3-MA, the chlorine peroxide (Cq) and the caspase inhibitor z-VAD-fmk to detect the change of the cell viability of the tumor, The apoptosis of the tumor cells was detected by the histone-DNA-ELISA and the Annexin V/ PI double-staining method. HK293 cell line was transfected with ATG5-si RNA to establish autophagy-deficient cells. MTT assay was used to detect the changes of cell activity of self-tophagy-deficient cells, and Annexin V/ PI double-staining method was used to detect the changes of apoptosis. Results: 1. The results of MTT and cell-colony experiments showed that the cell proliferation of glioma cells was significantly inhibited by the method of MTT and cell-colony, and the inhibitory effect was time-and concentration-dependent; Annexin V/ PI double-staining and histone-DNA-ELISA suggested that it was possible to induce the apoptosis of glioma cells, but the induction effect was not obvious; The expression of p-AKT was down-regulated by Western-Blot method, and the expression of autophagy-related protein ATG-5, LC-3B and Beclin-1 was up-regulated. The results of high performance liquid chromatography (HPLC) showed that the level of the neuroleptic amine in the cells could be slightly increased by the method of high performance liquid chromatography (HPLC). The results of ELISA and Annexin V/ PI double-staining showed that the apoptosis induction of glioma cells increased significantly. The results of Western-Blot show that the combination of the combination of the anti-autophagy-related protein ATG-5, LC-3B and Beclin-1 protein has no obvious effect on p-AKT. 3-MA and Cq inhibit the activity of glioma cells, but the inhibitory effect can be blocked by z-VAD-fmk, and 3-MA can enhance the inhibitory effect on the cell proliferation of glioblastoma. Both the histone DNA-linked immunosorbent assay (ELISA) and the Annexin V/ PI double-staining method can significantly enhance the induction of the apoptosis in the glioma cells, and the z-VAD-fmk can block the synergistic effect of the combination. The results of Annexin V/ PI double-staining showed that the induction of autophagy in the autophagy-deficient cells was significantly enhanced by the results of Annexin V/ PI double-staining. Conclusion: 1. Lirifamin is expected to be one of the candidate for the treatment of glioma, and has potential clinical application value. Neuralamine C6 enhances the killing of glioma cells and has a synergistic effect. The mechanism of the synergistic effect is to induce the autophagy reaction and the autophagy to inhibit the cell apoptosis while suppressing the proliferation of the glioma cells. and the neuropolyamine C6 is capable of inhibiting autophagy, restoring the apoptosis of the glioma cells caused by the palifosinine, and enhancing the cell killing effect of the palifosine on the glioma cells without relying on the AKT-m TOR signal conduction path.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R739.41
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相關(guān)期刊論文 前2條
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