蛋白激酶B抑制剂派立福辛联合神经酰胺C6抗人脑胶质瘤的机制研究
发布时间：2018-11-08 07:00
【摘要】：目的:观察蛋白激酶B抑制剂哌立福辛(perifosine)、神经酰胺C6(ceramide)对脑胶质瘤细胞增殖、凋亡、自噬等生物学行为的影响,探讨哌立福辛与神经酰胺C6联合用药的协同机制,为脑胶质瘤的靶向治疗提供理论依据。方法:1.对胶质瘤细胞予以不同浓度及时间的哌立福辛处理,运用MTT法及细胞集落实验检测肿瘤细胞增殖活性;组蛋白DNA ELISA法及Annexin V/PI双染法检测肿瘤细胞凋亡改变;Western-Blot法检测AKT1、p-AKT、p-S6、p-AMPKα、p-ACC以及ATG-5、LC-3B、Beclin-1的表达情况;高效液相色谱法检测胶质瘤细胞内神经酰胺的水平的变化。2.对胶质瘤细胞予以不同浓度及时间的神经酰胺C6处理,MTT法检测其对细胞活力的影响;神经酰胺C6、哌立福辛以及二者联合处理胶质瘤细胞,运用MTT法、Trypan blue实验检测肿瘤细胞活性改变;Western-Blot法检测ERK、p-AKT、p-S6、p-S6K、p-ERK以及ATG-5、LC-3B、Beclin-1的表达情况。3.运用自噬抑制剂3-MA、氯喹(Cq)和caspase抑制剂z-VAD-fmk对上述各组细胞予以干预,检测肿瘤的细胞活力变化;对胶质瘤细胞予以3-MA联合哌立福辛处理,MTT法检测肿瘤细胞活性,组蛋白DNA ELISA法及Annexin V/PI双染法检测肿瘤细胞凋亡改变;同样方法检测神经酰胺C6联合哌立福辛对胶质瘤细胞凋亡的影响。4.构建ATG5-si RNA转染HEK293细胞株建立自噬缺陷细胞;MTT法检测哌立福辛及神经酰胺C6对自噬缺陷细胞活性影响,Annexin V/PI双染法检测其凋亡改变。结果:1.MTT法和细胞集落实验结果显示哌立福辛显著抑制胶质瘤细胞增殖,其抑制效应呈时间和浓度依赖性;Annexin V/PI双染法及组蛋白DNA ELISA法提示哌立福辛能够诱导胶质瘤细胞凋亡,但诱导作用不明显;Western-Blot法检测到哌立福辛下调p-AKT的表达,上调自噬相关蛋白ATG-5、LC-3B、Beclin-1蛋白表达;高效液相色谱法结果显示哌立福辛能够轻度提高细胞内神经酰胺的水平。2.MTT法以及Trypan blue实验结果显示神经酰胺C6联合哌立福辛对胶质瘤细胞增殖抑制具有协同效应;组蛋白DNA ELISA法及Annexin V/PI双染法结果表明胶质瘤细胞凋亡诱导显著增加;Western-Blot法结果表明联合用药抑制自噬相关蛋白ATG-5、LC-3B、Beclin-1蛋白表达,对p-AKT影响不明显。3.自噬抑制剂3-MA,Cq显著抑制胶质瘤细胞活性,但抑制作用可被z-VAD-fmk阻断,3-MA可增强哌立福辛对胶质瘤的细胞增殖抑制效应;组蛋白DNA ELISA法及Annexin V/PI双染法显示神经酰胺C6可显著增强哌立福辛对胶质瘤细胞的凋亡诱导,z-VAD-fmk可阻断联合用药的协同作用。4.成功构建自噬缺陷细胞;哌立福辛对自噬缺陷细胞的增殖抑制作用显著增强,联合神经酰胺C6未见明显的协同效应;Annexin V/PI双染法检测结果显示哌立福辛对自噬缺陷细胞凋亡诱导作用明显增强。结论:1.哌立福辛有望作为胶质瘤分子靶向治疗治疗药物候选之一,具有潜在的临床应用价值。2.神经酰胺C6增强哌立福辛对胶质瘤细胞的杀伤作用,具有协同效应。3.协同作用的机制是派立福辛抑制胶质瘤细胞增殖的同时诱导自噬反应,自噬抑制细胞凋亡。而神经酰胺C6能够抑制自噬,恢复派立福辛导致的胶质瘤细胞凋亡作用,增强派立福辛对胶质瘤细胞的细胞杀伤作用而不依赖AKT-m TOR信号传导通路。
[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.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R739.41
本文编号：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.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R739.41
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