半胱氨酸萝卜硫素诱导人胶质母细胞瘤细胞凋亡和抑制侵润研究

发布时间：2018-03-24 20:21

本文选题：半胱氨酸萝卜硫素　切入点：ERK1/2　出处：《首都医科大学》2017年硕士论文

【摘要】：背景和目的:胶质母细胞瘤是一种常见的恶性程度极高的脑部肿瘤,标准治疗方案为手术配合放射性或化学疗法。因胶质母细胞瘤经常对常用化疗药物替莫唑胺(temozolomide,TMZ)产生耐药性,使得治疗后的患者存活期仅为15个月左右。因此,寻找一种高效低毒的化疗药物显得尤为重要。研究显示,十字花科植物能有效防治癌症的发生发展,萝卜硫素(Sulforaphane,SFN)是其天然提取成分。我们的前期结果表明SFN通过持续活化ERK1/2抑制胶质母细胞瘤细胞侵润,而半胱氨酸萝卜硫素(Sulforaphane-cysteine,SFN-Cys)是SFN类似物,也是SFN在人体内的代谢产物,具有更长的半衰期,可能能更有效的诱导细胞凋亡。本实验将研究SFN-Cys诱导胶质母细胞瘤U373MG和U87MG细胞凋亡的机制,为在临床上治疗胶质母细胞瘤患者提供理论支持。方法和结果:细胞增殖实验显示,SFN-Cys抑制胶质母细胞瘤U373MG和U87MG细胞增殖遵循剂量依赖关系。细胞形态学观察发现,SFN-Cys改变细胞形态,在SFN-Cys处理浓度为30μmol/L时,U373MG和U87MG细胞除了形态发生变化外,细胞数量也开始明显降低。我们用流式细胞术进一步验证了SFN-Cys对细胞凋亡的影响:SFN-Cys诱导U373MG和U87MG细胞凋亡遵循剂量依赖关系。Western blot实验显示,SFN-Cys活化ERK1/2遵循剂量依赖关系,上调Bax/Bcl-2比例和活化Caspase-3,而这些结果被ERK1/2抑制剂PD98059逆转。JC-1线粒体膜电位实验显示,SFN-Cys降低U373MG和U87MG细胞线粒体膜电位(mitochondrial membrane potential,MMP),而PD98059逆转了SFN-Cys诱导的MMP降低。结论:SFN-Cys活化ERK1/2并通过ERK1/2信号转导通路上调Bax/Bcl-2比例、活化Caspase-3等凋亡相关蛋白,最终诱导U373MG和U87MG细胞内源途径凋亡。这些发现预示SFN-Cys可能是一种优于SFN的抗人胶质母细胞瘤药物,尤其在耐药肿瘤中表现出巨大潜力。背景和目的:胶质母细胞瘤具有极强的侵袭能力,手术与放射性或化学治疗效果并未取得令人满意的结果,治疗后的患者存活期仅为15个月左右。我们的前期研究成果表明,十字花科植物提取成分萝卜硫素(Sulforaphane-cysteine,SFN-Cys)可以抑制胶质母细胞瘤和前列腺癌细胞侵润,这为治疗胶质母细胞瘤带来新的希望。而SFN类似物半胱氨酸萝卜硫素(Sulforaphane-cysteine,SFN-Cys)具有更突出的抗癌特点,可以穿过血脑屏障并有更长的半衰期。因此,本实验将进一步研究SFN-Cys抑制胶质母细胞瘤U373MG和U87MG细胞迁移和侵润的机制,为临床上治疗胶质母细胞瘤患者提供新药。方法和结果:细胞划痕实验和Transwell侵袭实验显示,SFN-Cys活化ERK1/2抑制SFN-Cys抑制胶质母细胞瘤U373MG和U87MG细胞迁移和侵润,并遵循剂量依赖关系。Western blot显示,SFN-Cys活化ERK1/2遵循剂量依赖关系,下调α-tubulin和Stathmin-1蛋白表达,上调p Stathmin-1(Ser 25)蛋白表达,而ERK1/2抑制剂可以逆转SFN-Cys对α-tubulin,Stathmin-1和p Stathmin-1(Ser 25)蛋白表达的调控。免疫荧光染色实验表明,SFN-Cys下调α-tubulin,破坏微管结构使其变为“鸟巢”状。激光共聚焦显微镜观察到,SFN-Cys虽下调Stathmin-1蛋白表达,但没有影响Stathmin-1与α-tubulin的结合能力。结论:SFN-Cys活化ERK1/2下调α-tubulin、Stathmin-1,上调p Stathmin-1(Ser 25)蛋白水平,最终抑制U373MG和U87MG细胞迁移和侵润。综上,SFN-Cys作为一种高效低毒的药物,将为胶质母细胞瘤患者带来新的希望。
[Abstract]:Background and objective: glioblastoma is a common malignant brain tumor, the standard treatment for radioactive or chemical therapy with surgery for glioblastoma. Often the commonly used chemotherapy drug temozolomide (temozolomide, TMZ) resistance, so that after the treatment of patients with survival was only 15 a month or so. Therefore, finding an effective chemotherapy drug is particularly important. Research shows that cruciferous plants can effectively prevent the development of cancer, sulforaphane (Sulforaphane, SFN) is the extraction of natural ingredients. Our early results show that the SFN through the persistent activation of ERK1/2 inhibited glioblastoma cell invasion run, and cysteine sulforaphen (Sulforaphane-cysteine, SFN-Cys) SFN analogues is SFN metabolites in the human body, has a longer half-life, could effectively induce cell apoptosis. The mechanism study of SFN-Cys induced U373MG glioma cells and the apoptosis of U87MG cells, for the treatment of glioblastoma patients in the clinic to provide theoretical support. Methods and results: cell proliferation assays demonstrated SFN-Cys inhibited the proliferation of U373MG glioma cells and U87MG cells following the dose dependent relationship. Cell morphology observation, cell morphology the change in SFN-Cys, SFN-Cys concentration is 30 mol/L, U373MG and U87MG cells in addition to morphological changes, the number of cells began to decrease. We use flow cytometry to further validate the effect of SFN-Cys on apoptosis induced by SFN-Cys: U373MG and U87MG cell apoptosis following the dose dependent relation of.Western blot experiment, SFN-Cys activation ERK1/2 follow the dose dependent up regulation of Bax/Bcl-2 ratio and activation of Caspase-3, and these results by ERK1/2 inhibitor PD98059 reversed the.JC-1 line According to the mitochondrial membrane potential and mitochondrial U373MG SFN-Cys experiment, reduce the membrane potential of U87MG cells (mitochondrial membrane potential, MMP), while PD98059 reversed the SFN-Cys induced decrease of MMP. Conclusion: the activation of SFN-Cys and ERK1/2 through ERK1/2 signal transduction pathway in the up regulation of Bax/Bcl-2 ratio, activation of Caspase-3 and other apoptosis related proteins, U373MG and U87MG cells induce endogenous pathway apoptosis. These findings indicate SFN-Cys may be glioblastoma against a drug is better than SFN, especially shows great potential in drug resistant tumors. Background and purpose: glioblastoma cell tumor with strong invasiveness, surgery and radioactive or chemical treatment did not achieve satisfactory results after treatment. Patient survival was only 15 months. The previous research results we show that cruciferous plant extract sulforaphane (Sulforaphane-cysteine, SFN-Cy S) can inhibit glioblastoma cells and prostate cancer cell invasion, which brings new hope for the treatment of glioblastoma. SFN analogs of cysteine sulforaphen (Sulforaphane-cysteine, SFN-Cys) with anticancer features more prominent, can cross the blood-brain barrier and has a long half-life. Therefore, the mechanism of the further study of SFN-Cys inhibition of U373MG glioma cells and U87MG cell migration and invasion, provide new drugs for the clinical treatment of patients with glioblastoma. Methods and results: display cell scratch assay and Transwell invasion assay, SFN-Cys activated ERK1/2 inhibited SFN-Cys inhibition of U373MG glioma cells and U87MG cell migration and invasion, and follow the dose dependent activation of SFN-Cys ERK1/2.Western blot display, follow the dose dependent expression of -tubulin alpha and Stathmin-1 protein, up-regulated P protein expression Stathmin-1 (Ser 25) Da, ERK1/2 inhibitors can reverse the SFN-Cys of -tubulin Stathmin-1 and P alpha, Stathmin-1 (Ser 25) protein expression. Immunofluorescence staining experiments showed that down-regulation of SFN-Cys alpha -tubulin, which becomes the "bird's nest" - destroy the microtubule structure. Laser confocal microscopy, SFN-Cys, Stathmin-1 protein expression was down regulated, but no with the ability to influence Stathmin-1 and alpha -tubulin. Conclusion: the activation of SFN-Cys ERK1/2 down-regulation of alpha -tubulin, Stathmin-1, Stathmin-1 (25 Ser) up-regulated P protein levels, ultimately inhibit U373MG and U87MG cell migration and invasion. In summary, SFN-Cys as a drug with high efficiency and low toxicity, will bring new hope to patients with glioblastoma.

【学位授予单位】：首都医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.41

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