自噬抑制剂3-MA增强顺铂对神经母细胞瘤化疗效果及其分子机制的研究

发布时间：2018-03-15 10:49

本文选题：神经母细胞瘤　切入点：自噬抑制剂3-MA　出处：《河北医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:研究通过抑制自噬,增强常规化疗药物顺铂对神经母细胞瘤SH-SY5Y治疗效果的可能性,并探究其可能的分子机制。方法:1将人神经母细胞瘤SH-SY5Y细胞株进行体外培养,采用不同浓度(0、1.25、2.5、5、10、20、40ug/ml)的顺铂分别处理处于对数生长期的神经母细胞瘤SH-SY5Y细胞24h、48h、72h,及不同浓度(0、1.25、2.5、5、10mM)的3-MA分别处理处于对数生长期的神经母细胞瘤SH-SY5Y细胞24h后,应用MTS比色法测定细胞的存活率,采用不同浓度(0、2.5、5、10、20ug/ml)的顺铂及联合应用自噬抑制剂3-MA(5mM)处理处于对数生长期的神经母细胞瘤SH-SY5Y细胞24h后,应用MTS比色法测定细胞的存活率,并分别计算单独应用顺铂及顺铂联合自噬抑制剂后顺铂的IC50值。2于普通光学显微镜下观察对照组、常规化疗药物顺铂(10ug/ml)组、自噬抑制剂3-MA(5mM)组、自噬抑制剂3-MA(5mM)联合常规化疗药物顺铂(10ug/ml)组细胞形态学上的改变。3用同样的方法处理细胞后,将四组处理后的细胞进行PI染色,在荧光显微镜下观察晚期凋亡及坏死细胞。4应用western blot方法分别检测上述四组细胞的自噬相关蛋白Beclin-1、LC3-Ⅱ/Ⅰ及凋亡相关蛋白Caspase-3的表达变化情况。实验数据应用SPSS 13.0统计软件进行统计学分析。计量资料均进行正态性及方差齐性检验,经检验后符合正态分布的计量资料采用均数±标准差(x±S)表示,不符合正态分布的计量资料采用中位数(四分位数间距)(M(P25~P75))表示。经检验符合正态分布且方差齐的多组均数的比较应用单因素方差分析(One-way ANOVA),多组间的两两比较采用SNK法(Student-Newman-Keuls);非正态分布的数据多组比较采用Kruskalwallis H检验,两两比较采用Mann-Whitney U检验。以α=0.05为检验标准,以p0.05作为差别显著性判断标准。结果:1经顺铂处理人神经母细胞瘤sh-sy5y细胞24小时后,对细胞活力影响较小,仅在顺铂浓度到达10ug/ml时,细胞存活率有所下降(p0.05)。顺铂作用48小时后,当顺铂浓度到达5ug/ml时,细胞活力下降(p0.01),当顺铂浓度到达10ug/ml时,细胞活力下降显著(p0.001)。顺铂作用72小时后,除浓度为1.25ug/ml组,其余各组细胞活力均下降显著,具有统计学意义(p0.05)。不同浓度的自噬抑制剂3-ma单独作用于神经母细胞瘤sh-sy5y细胞24h后,对细胞活力无明显影响,即使3-ma在最大浓度10mmol/l时,细胞存活率与空白对照组仍无统计学差异(p0.05)。单独应用顺铂及联合自噬抑制剂3-ma后顺铂的ic50值分别为10.9ug/ml和5.25ug/ml,联合自噬抑制剂后顺铂的ic50值降低了49%。2于光学显微镜下观察细胞形态学的改变,发现对照组细胞数目较多,均贴壁生长,细胞形态较一致,呈长梭形或多角形,体积较小,有聚集成团倾向,细胞代谢产物较少;自噬抑制剂处理组细胞数量无明显减少,但细胞凸起不明显,少量呈现椭圆形,顺铂处理组细胞数量明显减少,形态发生较明显变化,看不到长梭形状的细胞,胞质皱缩,多呈圆形或短椭圆形,聚集成团,自噬抑制剂联合顺铂组细胞数量减少更明显,且细胞分散,看不到明显成团细胞,培养皿内可见大量细胞产物及死亡细胞。3荧光显微镜下观察细胞的晚期凋亡及坏死情况发现,对照组细胞生长良好,未见晚期凋亡及坏死细胞,3-ma处理组晚期凋亡及坏死细胞较对照组无明显增多,顺铂处理组较对照组及3-ma处理组晚期凋亡及坏死细胞明显增多,顺铂联合3-ma处理组细胞的晚期凋亡及坏死率又明显高于单用顺铂组。4westernblot检测结果显示:对照组自噬及凋亡相关蛋白均处于基础水平,应用3-ma抑制自噬后,检测到自噬相关蛋白表达下降,但凋亡相关蛋白caspase-3表达并未见明显增高;顺铂处理神经母细胞瘤sh-sy5y细胞24h后,与对照组相比自噬相关蛋白beclin-1、lc3-Ⅱ/Ⅰ及凋亡相关蛋白caspase-3的表达均升高,加入自噬抑制剂后,自噬相关蛋白Beclin-1、LC3-Ⅱ/Ⅰ的表达较单独应用顺铂组明显下降,但凋亡相关蛋白Caspase-3的表达较单独应用顺铂组明显升高,差异有统计学意义(P0.05)。结论:1顺铂能够在体外抑制神经母细胞瘤SH-SY5Y细胞的增值,且在一定的浓度范围内这种抑制作用具有时间、剂量依赖性;2单独应用自噬抑制剂3-MA 24小时,在一定的浓度范围内无明显抑制增值作用,即单独抑制自噬并不能有效抑制神经母细胞瘤的增殖;3顺铂联合自噬抑制剂3-MA后,可降低顺铂的IC50值,起化疗增敏作用;4顺铂诱导神经母细胞瘤SH-SY5Y细胞发生凋亡的同时,自噬作用增强,且自噬在细胞凋亡过程中起保护作用;5抑制自噬可以加强顺铂诱导神经母细胞瘤SH-SY5Y细胞凋亡的作用,增强其化疗效果,提示自噬抑制剂可提高常规化疗药物顺铂对神经母细胞瘤的治疗效果,较单纯常规化疗更有价值,为神经母细胞瘤的临床治疗提供了新的思路和方法。
[Abstract]:Objective: To study the inhibition of autophagy, enhance the possibility of conventional chemotherapy drug cisplatin on neuroblastoma SH-SY5Y treatment, and to explore its possible molecular mechanisms. Methods: 1 human neuroblastoma cell line SH-SY5Y were cultured in vitro, with different concentrations of cisplatin (0,1.25,2.5,5,10,20,40ug/ml) treatment in neuroblastoma SH-SY5Y cells 24h, logarithmic growth phase and different concentrations of 48h, 72h (0,1.25,2.5,5,10mM) were treated with 3-MA in neuroblastoma SH-SY5Y cells in logarithmic growth phase after 24h, colorimetric determination of cell survival rate by MTS, using different concentration of cisplatin (0,2.5,5,10,20ug/ml) and the combined application of autophagy inhibitor 3-MA (5mM) in the treatment of SH-SY5Y cells in logarithmic growth phase after 24h, colorimetric determination of cell survival rate by MTS, and calculated the separate application of cisplatin and cisplatin Combined with autophagy inhibitor after cisplatin IC50 value of.2 in ordinary optical microscope in control group, conventional chemotherapy drug cisplatin (10ug/ml) group, autophagy inhibitor 3-MA (5mM) group, autophagy inhibitor 3-MA (5mM) combined with conventional chemotherapy drug cisplatin (10ug/ml) group of cell morphological changes of.3 cells treated with the same method. The four groups of the treated cells were stained with PI, observed late apoptosis and necrosis were detected using western.4 blot of the four group of the autophagy related protein Beclin-1 in the fluorescence microscope, the expression of LC3- II / I and apoptosis related protein Caspase-3. The experimental data using SPSS 13 statistical software for statistical analysis. The measurement data were normality and homogeneity of variance test, after testing with normal distribution measurement data by the mean and standard deviation (x + S) said, does not conform to the normal distribution and measurement The median (four percentile interval) (M (P25~P75)). After testing in line with the analysis of the normal distribution and homogeneity of multiple mean comparison using single factor variance (One-way, ANOVA) - between 22 groups were compared with SNK method (Student-Newman-Keuls); non normal distribution data sets compared with Kruskalwallis H test, 22 compared with Mann-Whitney U test. A =0.05 test, with P0.05 as the difference significant criteria. Results: 1 after treatment with cisplatin in human neuroblastoma SH-SY5Y cells after 24 hours, the cell viability is less affected, only reached 10ug/ml in cisplatin concentration, cell survival rate decreased (P0.05). 48 hours after cisplatin, when cisplatin concentration reached 5ug/ml, decreased cell viability (P0.01), when the cisplatin concentration reached 10ug/ml, cell viability decreased significantly (p0.001). The effect of cisplatin for 72 hours, in addition to a concentration of 1.25 Ug/ml group, cell viability decreased significantly in other groups, with statistical significance (P0.05). Autophagy inhibitors with different concentrations of 3-mA alone in neuroblastoma SH-SY5Y cells after 24h had no obvious effect on cell viability, even if the 3-mA in the maximum concentration of 10mmol/l, the cell survival rate was no significant difference with the control group (P0.05). Cisplatin alone and in combination with autophagy inhibitor 3-mA after cisplatin IC50 values were 10.9ug/ml and 5.25ug/ml, IC50 reduced 49%.2 in cisplatin under optical microscope to observe the cell morphology changes value combined with autophagy inhibitor, was found in the control group had more number of cells were adherent growth, cell morphology was similar to that of spindle or polygonal, small volume, has aggregated tendency, cell metabolism is less; the number of cells without the autophagy inhibitor treatment group decreased significantly, but the cell protrusion is not obvious, a small present oval The shape, cisplatin treatment group significantly decreased, morphology significantly changed, can not see the long spindle shaped cells, cytoplasmic shrinkage, round or oval, together, the number of cell autophagy inhibitor combined with cisplatin group decreased significantly, and the cells dispersed, do not see a significant cluster of cells, culture there were a lot of dish products and death of.3 cells were observed under fluorescence microscope late apoptosis and necrosis of cells that controls cell growth is good, no late apoptotic and necrotic cells, 3-mA treatment group late apoptosis and necrosis than in control group significantly increased, cisplatin group compared with control group and 3-mA treatment group and late apoptosis necrotic cells increased significantly, cisplatin combined with 3-mA treatment group advanced cell apoptosis and necrosis rate was significantly higher than that of cisplatin group.4westernblot test results show that: the group of autophagy and apoptosis related protein At the basic level, the application of 3-mA after the inhibition of autophagy, detected the expression of autophagy related protein decreased, but the expression of apoptosis related protein caspase-3 was significantly increased; cisplatin treated neuroblastoma SH-SY5Y cells after 24h, compared with the control group the expression of autophagy related protein beclin-1, lc3- II / I and apoptosis related protein caspase-3 increased significantly. After joining the autophagy inhibitor, autophagy related protein Beclin-1, LC3- II / I. expression compared with cisplatin alone group decreased significantly, but the expression of apoptosis related protein Caspase-3 compared with cisplatin alone group was significantly increased, the difference was statistically significant (P0.05). Conclusion: 1 cisplatin can inhibit neuroblastoma SH-SY5Y cells in vitro added. And this in a certain concentration range inhibition is time dose dependent; 2 separate application of autophagy inhibitor 3-MA for 24 hours, in a certain concentration range without obvious The growth inhibition effect, which alone can not effectively inhibit autophagy and inhibit neuroblastoma proliferation; 3 cisplatin combined with autophagy inhibitor 3-MA, cisplatin can reduce the IC50 value on chemotherapy sensitization; neuroblastoma cell apoptosis induced by cisplatin 4 SH-SY5Y at the same time, enhance the apoptosis and autophagy plays a protective role. In the process of cell apoptosis; 5 inhibition of autophagy can enhance cisplatin induced apoptosis of neuroblastoma SH-SY5Y cells and enhance the effect of chemotherapy, suggesting that autophagy inhibitors may improve the therapeutic effect of conventional chemotherapy drug cisplatin on neuroblastoma cells, compared with conventional chemotherapy is more valuable, to provide new ideas and methods for clinical treatment of nerve blastomas.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.4

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