白藜芦醇抗人脑胶质瘤作用及机制研究

发布时间：2018-05-31 03:27

本文选题：脑胶质瘤 + 白藜芦醇　；参考：《武汉大学》2014年博士论文

【摘要】：脑胶质瘤是原发性颅内肿瘤中最常见的恶性肿瘤。虽然近年来神经外科领域已得到长足发展,但即使是最现代的神经外科技术,也难以对脑胶质瘤做到病理学上的完全切除。脑胶质瘤的生物学行为主要表现为局部扩散及高度侵袭,因而胶质瘤术后复发率高。临床上迫切需要有效的辅助治疗方法,以改善脑胶质瘤的预后。白藜芦醇(resveratrol, Res)是从虎杖、藜芦、葡萄等天然植物中提取出来的多酚化合物,具有调节脂质代谢、抑制血小板聚集、保护心血管、抗炎等多种生物学活性。近年研究发现,白藜芦醇对乳腺癌、前列腺癌、皮肤癌、胃癌等多种肿瘤细胞的生长均具有显著的抑制作用,尤其是对脑肿瘤而言,血脑屏障对白藜芦醇具有良好的通透性,是一种非常有前景的天然抗肿瘤药物。第一部分目的探讨白藜芦醇对脑胶质瘤细胞增殖的抑制作用及其可能的机制。方法首先将不同浓度的白藜芦醇(0、25、50、100μ M)作用于脑胶质瘤U87细胞和U251细胞,倒置显微镜定性观察白藜芦醇对脑胶质瘤细胞形态的影响,台盼蓝拒染法及MTT增殖抑制试验定量检测白藜芦醇对脑胶质瘤细胞生长的抑制作用,然后再用RT-PCR及Western blot检测EGFR、LRIG1、磷酸化Akt及ERk核酸水平以及蛋白表达水平的变化。结果经不同浓度白藜芦醇分别处理脑胶质瘤U87细胞及U251细胞后,细胞活力逐渐下降,贴壁能力差,细胞开始变圆,细胞体积变小,一部分细胞开始悬浮,与瓶底分离。台盼蓝拒染法及MTT增殖抑制试验表明,白藜芦醇能抑制脑胶质瘤U251细胞及U87细胞的生长,其作用强度呈时间及剂量依赖性。RT-PCR及western blot实验结果发现,经白藜芦醇处理后脑胶质瘤细胞中EGFR蛋白表达水平下降,而其负性调节蛋白LRIG1蛋白的表达升高,白藜芦醇对ERK磷酸化的表达无影响,但能够明显抑制Akt磷酸化。结论白藜芦醇能够抑制脑胶质瘤细胞的增殖,其机制可能主要是通过抑制PI3K/Akt信号转导通路来发挥对脑胶质瘤细胞增殖的抑制作用。第二部分目的研究白藜芦醇诱导脑胶质瘤细胞凋亡的作用及其具体机制。方法用DNA琼脂糖凝胶电泳及Annexin V-FITC凋亡试剂盒检测白藜芦醇对脑胶质瘤细胞凋亡的诱导作用,进一步用Rhodamine123荧光染料染色后经流式细胞仪检测,再用Western blot检测内源性凋亡途径相关蛋白Cyto C及caspase-9的表达。结果经25μM、50μM、100μM浓度的白藜芦醇处理U87细胞后DNA琼脂糖凝胶电泳呈现凋亡细胞的梯级(ladder)格局,而对照组细胞DNA无断裂现象；Annexin V-FITC凋亡试剂盒检测也发现白藜芦醇能明显诱导U87细胞发生凋亡；经白藜芦醇处理后,U87细胞的线粒体膜电位明显降低,这一作用呈剂量依赖性。Western blot检测发现Cyto C及caspase-9蛋白的表达增强。结论白藜芦醇可以促进Cyto C释放及Caspase-9蛋白表达,通过激活内源性细胞凋亡途径来发挥抗肿瘤作用。第三部分目的探讨白藜芦醇对脑胶质瘤细胞自噬的诱导作用及可能的作用机制。方法用不同浓度白藜芦醇处理脑胶质瘤U87细胞24h后,在电子显微镜下观察细胞形态的变化。应用半定量RT-PCR及Realtime PCR分析白藜芦醇对自噬相关基因LC-3、Beclin-1、VPS34基因表达的影响。Western blot检测白藜芦醇对LC-3、Beclin-1、VPS34蛋白表达水平的影响。结果在电子显微镜下可以观察到典型的自噬性空泡的出现,而对照组细胞未出现明显自噬空泡。白藜芦醇处理前U87细胞中LC-3、Beclin-1、VPS34基因为低表达,经25μM、50μM,100μM浓度的白藜芦醇处理24h后,LC-3、Beclin-1、VPS34基因mRNA的表达明显增强。白藜芦醇作用后可明显升高LC-3蛋白的含量,Beclin-1、VPS34的表达也逐渐升高。结论自噬过程参与了白藜芦醇诱导的U87细胞死亡,LC-3、Beclin-1、VPS34基因表达上调是其诱导自噬的可能机制。第四部分目的脑胶质瘤的恶性生物学行为主要表现为局部扩散及高度侵袭转移,本部分研究白藜芦醇对脑胶质瘤细胞侵袭转移的抑制作用及可能机制。方法通过细胞划痕实验研究白藜芦醇对脑胶质瘤细胞迁移运动能力的影响,Transwell侵袭小室实验研究白藜芦醇对脑胶质瘤细胞侵袭和转移能力的影响,RT-PCR及Western Blot从mRNA表达及蛋白表达水平检测经白藜芦醇作用后,脑胶质瘤细胞中MMP-2、MMP-9、TIMP-1、TIMP-2蛋白水平的变化。结果白藜芦醇能明显抑制脑胶质瘤细胞的迁移运动能力及侵袭、转移能力；能够降低脑胶质瘤U87细胞中MMP-2、MMP-9的表达,使TIMP-1、TIMP-2的表达提高,且MMP-2和MMP-9的表达随着白黎芦醇浓度的增加而呈现下降的趋势。结论白黎芦醇可能通过抑制MMP-2及MMP-9的表达并调节TIMP-2/MMP-2,TIMP-1/MMP-9的动态平衡来抑制脑胶质瘤细胞的迁移和侵袭。本课题研究表明白藜芦醇可以通过对脑胶质瘤细胞增殖的抑制、诱导细胞凋亡及自噬和抑制细胞侵袭转移等四个方面发挥其抗肿瘤作用,本研究为白藜芦醇用于脑胶质瘤的临床治疗提供了新的理论依据。
[Abstract]:Glioma is the most common malignant tumor in primary intracranial tumors. Although the field of Department of neurosurgery has developed rapidly in recent years, even the most modern department of Neurosurgery technique is difficult to complete complete pathological excision of glioma. The biological behavior of glioma is mainly characterized by local diffusion and high invasion. The recurrence rate of glioma after operation is high. It is urgent to use effective adjuvant therapy to improve the prognosis of brain glioma. Resveratrol (Res) is a polyphenol compound extracted from natural plants such as Polygonum cuspidatum, veratris, grape and other natural plants, which can regulate lipid metabolism, inhibit platelet aggregation, protect cardiovascular, anti-inflammatory and so on. In recent years, it has been found that resveratrol has a significant inhibitory effect on the growth of many tumor cells, such as breast cancer, prostate cancer, skin cancer and gastric cancer. Especially for brain tumors, the blood brain barrier has a good permeability to resveratrol. It is a very promising natural antitumor drug.
The first part is to investigate the inhibitory effect of resveratrol on the proliferation of glioma cells and its possible mechanism. Methods first, the resveratrol (0,25,50100) of resveratrol (resveratrol) of different concentrations was used to act on the U87 cells and U251 cells of glioma, and the effect of resveratrol on the morphology of the glioma cells was observed by inverted microscope, and trypan blue staining method was used to determine the effect of resveratrol on the cell morphology of brain glioma. The inhibitory effect of resveratrol on the growth of glioma cells was detected by MTT proliferation inhibition test and then RT-PCR and Western blot were used to detect the changes of EGFR, LRIG1, phosphorylated Akt, ERk nucleic acid level and protein expression level. Results after different concentrations of resveratrol in the glioma U87 and U251 cells, the cells live The force gradually declined, the adhesion ability was poor, the cell began to circle, the cell volume became smaller, some cells began to suspend and separated from the bottle bottom. Trypan blue exclusion assay and MTT proliferation inhibition test showed that resveratrol could inhibit the growth of U251 and U87 cells in glioma, and its action intensity was time and dose dependent.RT-PCR and Western blot experiments The results showed that the expression level of EGFR protein in the glioma cells after resveratrol treatment decreased, but the expression of its negative regulatory protein LRIG1 protein increased, and resveratrol had no effect on the expression of ERK phosphorylation, but could obviously inhibit the phosphorylation of Akt. Conclusion resveratrol can inhibit the proliferation of glioma cells, and the mechanism may be main. It inhibits the proliferation of glioma cells by inhibiting PI3K/Akt signaling pathway.
The second part aims to study the effect of resveratrol on the apoptosis of glioma cells induced by resveratrol and its specific mechanism. Methods the induction of resveratrol on the apoptosis of glioma cells was detected by DNA agarose gel electrophoresis and Annexin V-FITC apoptosis kit, and then detected by flow cytometry after staining with Rhodamine123 fluorescent dye, and then Wes The expression of endogenous apoptotic pathway related protein Cyto C and caspase-9 was detected by tern blot. The results showed the cascade (ladder) pattern of apoptotic cells by DNA agarose gel electrophoresis after 25 u M, 50 mu M, and 100 u M concentration of resveratrol. The apoptosis of U87 cells could be induced by resveratol. After resveratrol treatment, the mitochondrial membrane potential of U87 cells decreased significantly. The effect of.Western blot detection showed that the expression of Cyto C and caspase-9 protein was enhanced.
Conclusion resveratrol can promote the release of Cyto C and the expression of Caspase-9 protein, and play an anti-tumor role by activating the endogenous apoptotic pathway.
The third part aims to explore the induction and mechanism of resveratrol on autophagy in glioma cells. Methods the morphologic changes were observed under the electron microscope after treating glioma U87 cell 24h with resveratrol with different concentrations. The autophagy related gene LC-3 was analyzed by semi quantitative RT-PCR and Realtime PCR. The effect of Beclin-1, VPS34 gene expression on.Western blot to detect the effect of resveratrol on the expression of LC-3, Beclin-1, and VPS34 protein. Results the typical autophagic vacuoles can be observed under the electron microscope, while the cells in the control group have no obvious autophagic vacuoles. LC-3, Beclin-1, VPS34 base in the U87 cells before resveratrol treatment The expression of LC-3, Beclin-1, VPS34 gene mRNA was obviously enhanced after low expression of resveratrol at 25 M, 50 M and 100 micron M. The content of LC-3 protein was obviously increased after resveratrol action, and the expression of Beclin-1 and VPS34 increased gradually. Conclusion autophagy was involved in the death of resveratrol induced U87 cells. The up-regulated expression is a possible mechanism for its induction of autophagy.
In the fourth part, the malignant biological behavior of brain glioma is mainly characterized by local diffusion and high invasion and metastasis. This part studies the inhibitory effect of resveratrol on the invasion and metastasis of glioma cells and its possible mechanism. Methods the effect of resveratrol on the migration and movement of brain glioma cells was studied by cell scratch test, Transwel The effect of resveratrol on the invasion and metastasis of glioma cells, and the changes in the level of MMP-2, MMP-9, TIMP-1, TIMP-2 protein in glioma cells after resveratrol were detected by resveratrol on the invasion and metastasis of glioma cells in the L invasion experiment. The results showed that resveratrol could significantly inhibit glioma cells in the brain glioma cells after resveratrol action. Migration ability, invasion and metastasis ability, can reduce the expression of MMP-2 and MMP-9 in glioma U87 cells, improve the expression of TIMP-1 and TIMP-2, and the expression of MMP-2 and MMP-9 decreases with the increase of the concentration of resveratol. Conclusion resveratol may inhibit the expression of MMP-2 and MMP-9 and regulate TIMP-2/MMP-2, TIMP The dynamic balance of -1/MMP-9 inhibits the migration and invasion of glioma cells.
This study shows that veratrol can inhibit the proliferation of glioma cells, induce apoptosis, autophagy and inhibit cell invasion and metastasis in four aspects. This study provides a new theoretical basis for the clinical treatment of resveratrol for brain glioma.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.41

【参考文献】