姜黄素靶向调控HSP90及对胶质瘤细胞增殖和侵袭作用的研究

发布时间：2018-07-05 18:41

本文选题：胶质瘤 + HSP90　；参考：《山东大学》2016年博士论文

【摘要】：目的本研究对胶质瘤组织及正常脑组织中HSP90蛋白的表达、细胞增殖和凋亡进行测定,分析HSP90与胶质瘤患者临床病理特征的相关性及其对细胞增殖和凋亡的影响,以探讨姜黄素靶向调控HSP90抑制胶质瘤细胞增殖和侵袭的作用机制,旨在为胶质瘤治疗提供理论基础及新的思路,同时也为寻找新的HSP90抑制剂治疗胶质瘤奠定基础。方法(1)采集50例神经胶质瘤组织样本,其中男性28例,女性22例。年龄在23-78岁之间,平均为37.55±11.2岁；所有胶质瘤患者中,WHO I级6例,II级8例,III级17例,IV级19例；同时设立经CT和MRI等影像学检测证实无任何肿瘤性疾病的10例由于外伤或者脑出血而进行颅内减压的正常脑组织样本作为对照。运用western blot法分别检测上述样本中HSP90蛋白的表达情况,分析HSP90的表达与临床病理分级的相关性。(2)将HSP90 siRNA转染胶质瘤T98G细胞用以抑制其表达,荧光定量PCR检测转染后细胞中HSP90 mRNA的表达情况；MTT法检测转染后细胞的增殖情况；流式细胞术检测转染后细胞的凋亡情况。(3)体外malachite green-molybdate显色反应以及ATP-琼脂糖凝胶结合实验检测姜黄素对HSP90的抑制活性,同时分别运用MTT法、Transwell小室法检测姜黄素对T98G细胞增殖和侵袭的影响,此外通过western blot法检测姜黄素对基质金属蛋白9(MMP-9)以及HSP90表达的影响。结果(1)在胶质瘤组织中HSP90蛋白表达量显著高于其在正常脑组织中的表达(P0.01),且随着病理级别的增高其表达量呈上升趋势；运用Spearman软件分析胶质瘤患者临床病理分级与HSP90蛋白表达的相关性,结果为：HSP90蛋白表达的相关系数r=0.713(P0.001),表明胶质瘤组织中HSP90蛋白表达与临床病理分级呈正相关,即在高级别病例组中高表达,在低级别病例组中低表达。(2)随着时间的延长,HSP90 siRNA作用后T98G细胞的增殖能力逐渐下降。当作用第3、4、5、6天时,siHSP90组T98G细胞的增殖活性较对照组均有下降,其中第4、5、6天时降低幅度显著,差异具有显著性统计学意义(P0.05)；对照组细胞的凋亡率为(8.35±1.27)%,siHSP90组细胞的凋亡率为(17.88±0.13)%,两组相比差异具有高度显著性统计学意义(P0.01)。(3)姜黄素能够抑制HSP90重组蛋白的ATPase活性,其活性的IC50值为83.3μ/mol;姜黄素能够抑制HSP90蛋白与ATP-琼脂糖凝胶株结合,且这种结合抑制作用随姜黄素浓度的增高而增强；T98G细胞增殖能力随着姜黄素浓度的增高而逐渐降低,即姜黄素浓度越高其抑制T98G细胞增殖的作用越明显,呈剂量依赖性；T98G细胞的侵袭能力随着姜黄素浓度的增高而逐渐降低；MMP-9蛋白的表达量随着姜黄素浓度的增高而逐渐降低,且呈剂量依赖性,而HSP90蛋白的表达则未发生明显变化。结论姜黄素能够显著抑制胶质瘤细胞增殖以及侵袭能力,其机制可能是通过抑制HSP90 ATPase活性致使其下游顾客蛋白MMP-9的表达量下降有关。
[Abstract]:Objective to investigate the expression of HSP90 protein, cell proliferation and apoptosis in glioma and normal brain tissues, and to analyze the relationship between HSP90 and clinicopathological features of glioma and its effect on cell proliferation and apoptosis. In order to explore the mechanism of curcumin targeting regulation of HSP90 on the proliferation and invasion of glioma cells, the aim is to provide a theoretical basis and new ideas for the treatment of glioma, and to find a new HSP90 inhibitor for the treatment of glioma. Methods (1) 50 cases of gliomas were collected, including 28 males and 22 females. The age ranged from 23 to 78 years old (mean 37.55 卤11.2 years). Among all the glioma patients, there were 6 cases of WHO grade I, 8 cases of grade II, 17 cases of grade III and 19 cases of grade IV. At the same time, 10 cases of normal brain tissues with intracranial decompression due to trauma or intracerebral hemorrhage, which were confirmed by CT and MRI, were used as control group. The expression of HSP90 protein was detected by western blot method, and the correlation between HSP90 expression and clinicopathological grade was analyzed. (2) HSP90 siRNA was transfected into glioma T98G cells to inhibit the expression of HSP90 protein. The expression of HSP90 mRNA in transfected cells was detected by fluorescence quantitative PCR and the proliferation of transfected cells was detected by MTT assay. Flow cytometry was used to detect the apoptosis of transfected cells. (3) in vitro malachite green-molybdate reaction and ATP-agarose gel binding assay were used to detect the inhibitory activity of curcumin on HSP90. The effects of curcumin on the proliferation and invasion of T98G cells were detected by MTT assay and transwell chamber assay respectively. The effects of curcumin on the expression of matrix metalloprotein 9 (MMP-9) and HSP90 were detected by western blot assay. Results (1) the expression of HSP90 protein in glioma tissues was significantly higher than that in normal brain tissues (P0.01), and the expression of HSP90 protein increased with the increase of pathological grade. The correlation between the expression of HSP90 protein and the clinicopathological grade of glioma was analyzed by Spearman software. The results showed that the expression of HSP90 protein in glioma tissues was positively correlated with the clinicopathologic grade, and the correlation coefficient of the expression of HSP90 protein was rn0.713 (P0.001). That is, high expression in high grade cases, and low expression in low grade cases. (2) the proliferation ability of T98G cells decreased gradually with time prolonging after HSP90 siRNA treatment. The proliferation activity of T98G cells in HSP90 group was significantly lower than that in the control group on the 3rd day (P 0.05), and significantly decreased on the 4th day (P 0.05), while on the 6th day after treatment, the proliferation activity of T98G cells in HSP90 group was significantly lower than that in the control group (P 0.05). The apoptosis rate of the control group was (8.35 卤1.27) and that of the control group was (17.88 卤0.13). The difference between the two groups was statistically significant (P0.01). (3) curcumin could inhibit the ATPase activity of HSP90 recombinant protein. The IC50 of HSP90 protein was 83.3 渭 / mol. Curcumin could inhibit the binding of HSP90 protein to ATP-agarose gel strain, and the inhibitory effect increased with the increase of curcumin concentration, the proliferation ability of T98G cells decreased with the increase of curcumin concentration. The higher the curcumin concentration, the more obvious the inhibitory effect of curcumin on the proliferation of T98G cells. The invasion ability of T98G cells decreased with the increase of curcumin concentration, and the expression of MMP-9 protein decreased with the increase of curcumin concentration. The expression of HSP90 protein did not change in a dose-dependent manner. Conclusion curcumin can significantly inhibit the proliferation and invasion of glioma cells by inhibiting the activity of HSP90 ATPase resulting in the decrease of MMP-9 expression in the downstream of glioma cells.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R739.41

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