【作者】 李晨；

【导师】 葛鹏飞；

【作者基本信息】 吉林大学 ， 外科学（专业学位）， 2022， 硕士

【摘要】 研究背景:脑胶质瘤是最常见的原发性颅内恶性肿瘤,其标准疗法主要是手术切除联合术后放化疗。替莫唑胺(Temozolomide,TMZ)凭借其高口服生物利用度、亲脂性和小尺寸的优点,具有穿越血脑屏障的能力,是高级别胶质瘤化疗的一线药物。然而多数胶质瘤患者的预后较差,即使经过标准治疗,中位生存期仅为14.6个月,这与胶质瘤的耐药性相关。轻链亚基(xCT,SLC7A11)是Xc~-系统的重要组成单位,近年来研究发现xCT在各种癌症的发生发展、侵袭转移、铁死亡和耐药性中起着重要作用,因此,通过抑制xCT(SLC7A11)的表达水平来减少胶质瘤细胞的替莫唑胺耐药性有着重要意义。水飞蓟宾(Silibinin)是从菊利植物水飞蓟(Silybum marianum)果实中提取分离而得的一种黄酮类天然化合物,已被广泛用于治疗和预防多种类型的肝胆疾病,同时研究表明,水飞蓟宾对多种类型的肿瘤细胞具有抑制作用,并且可以提高胶质瘤细胞对替莫唑胺的敏感性,但目前还不清楚水飞蓟宾影响胶质瘤细胞替莫唑胺耐药性的机制。探讨xCT(SLC7A11)在复发脑胶质瘤及替莫唑胺耐药胶质瘤细胞中的表达特点及水飞蓟宾对其表达的调控作用,有助于为改善胶质瘤患者的预后提供理论基础。目的:采用随机选取并分组的脑胶质瘤临床病理标本和人胶质瘤细胞系研究其xCT(SLC7A11)表达变化的特点,并探讨水飞蓟宾在胶质瘤及替莫唑胺耐药胶质瘤细胞中对xCT表达的调控作用。方法:1、Western Blotting检测随机选取的4组脑胶质瘤和瘤旁组织样本中xCT(SLC7A11)的表达变化差异。2、Western Blotting检测随机选取的8个不同恶性程度脑胶质瘤组织样本中xCT(SLC7A11)的表达变化差异。3、体外培养人U87和U373细胞并构建U87和U373的TMZ耐药细胞株(U87TR和U373TR),Western Blotting检测U373和U373TR中xCT(SLC7A11)的表达变化差异;半胱氨酸和GSH检测试剂盒检测U87和U87TR细胞中的半胱氨酸和GSH含量。4、通过MTT法测定不同浓度水飞蓟宾对胶质瘤细胞的抑制作用;Western Blotting检测水飞蓟宾对人U87和U87TR细胞作用不同时间导致xCT(SLC7A11)的表达变化差异。5、乳酸脱氢酶释放实验检测低浓度水飞蓟宾对TMZ诱导U87细胞死亡的影响;Western Blotting检测TMZ及低浓度水飞蓟宾联合TMZ对U87细胞中xCT(SLC7A11)的表达影响。结果:1、脑胶质瘤组织中xCT(SLC7A11)的表达水平明显高于瘤旁组织。2、脑胶质瘤组织中xCT(SLC7A11)的表达水平随着恶性程度越增高而呈上升趋势。3、U373TR细胞中xCT(SLC7A11)的表达水平高于U373细胞,并随着U373TR细胞中替莫唑胺耐药浓度的升高而呈上升趋势;U87TR细胞中半胱氨酸和GSH含量高于U87细胞。4、水飞蓟宾对胶质瘤细胞有增殖抑制作用并且可以抑制U87和U87TR细胞中xCT(SLC7A11)的表达水平。5、低浓度水飞蓟宾联合应用TMZ提高了TMZ对U87细胞的杀伤效果,并抑制TMZ诱导的xCT(SLC7A11)表达水平的上升。结论:1.xCT(SLC7A11)在脑胶质瘤组织中高表达,并随着脑胶质瘤组织恶性程度的增高呈表达上升趋势。2.xCT(SLC7A11)在复发和替莫唑胺耐药胶质瘤细胞中高表达。3.水飞蓟宾通过抑制xCT(SLC7A11)的表达发挥对抗胶质瘤细胞活性和替莫唑胺耐药性的作用。4.低浓度水飞蓟宾联合应用替莫唑胺通过抑制xCT(SLC7A11)的表达减少胶质瘤细胞的替莫唑胺耐药性并发挥协同杀伤作用。更多还原

【Abstract】 Background:Glioma is the most common primary intracranial malignant tumor,and its standard treatment is mainly surgical resection combined with postoperative radiotherapy and chemotherapy.Temozolomide(TMZ),with its high oral bioavailability,lipophilicity,and small size,has the ability to cross the blood-brain barrier and is the first-line drug for high-grade glioma chemotherapy.However,the prognosis of most glioma patients is poor,even after standard treatment,the median survival time is only 14.6 months,which is related to the drug resistance of glioma.The light-chain subunit(xCT,SLC7A11)is an important component of the X_C~-system.In recent years,studies have found that xCT plays an important role in the occurrence and development,invasion and metastasis,ferroptosis and drug resistance of various cancers.Therefore,it is of great significance to reduce the temozolomide resistance of glioma cells by inhibiting the expression level of xCT(SLC7A11).Silibinin is a flavonoid natural compound extracted and isolated from the fruit of the chrysanthemum plant,Silybum marianum.It has been widely used in the treatment and prevention of various types of hepatobiliary diseases.At the same time,studies have shown that silibinin has inhibitory effects on various types of tumor cells,and can improve the sensitivity of glioma cells to temozolomide,but it is still unclear how silibinin affects the mechanisms of temozolomide resistance in glioma cells.To investigate the expression characteristics of xCT(SLC7A11)in recurrent glioma and temozolomide-resistant glioma cells and the regulation of silibinin on its expression,which will help to provide a theoretical basis for improving the prognosis of glioma patients.Objective:Randomly selected and grouped glioma clinicopathological specimens and human glioma cell lines were used to study the characteristics of xCT(SLC7A11)expression changes.In addition,we explored the regulatory effect of silibinin on xCT expression in glioma and temozolomide-resistant glioma cells.Methods:1.Western Blotting detected the differences in the expression of xCT(SLC7A11)in randomly selected 4 groups of glioma and paratumor tissue samples.2.Western Blotting detected the differences in the expression of xCT(SLC7A11)in 8 randomly selected glioma tissue samples with different degrees of malignancy.3.Human U87 and U373 cells were cultured in vitro and used to construct TMZ-resistant cell lines(U87TR and U373TR)of U87 and U373.Western Blotting was used to detect the differences in the expression of xCT(SLC7A11)in U373 and U373TR;Cysteine and GSH detection kits detected cysteine and GSH content in U87and U87TR cells.4.Determination of the inhibitory effect of different concentrations of silibinin on glioma cells by MTT assay;Western Blotting detected differences in the expression of xCT(SLC7A11)induced by silibinin on human U87 and U87TR cells at different times.5.LDH release assay was used to detect the effect of low-concentration silibinin on TMZ-induced U87 cell death;Western Blotting was used to detect the effect of TMZ and low-concentration silibinin combined with TMZ on the expression of xCT(SLC7A11)in U87 cells.Results:1.The expression level of xCT(SLC7A11)in glioma tissue was significantly higher than that in adjacent tissue.2.The expression level of xCT(SLC7A11)in glioma tissue increased with the increasing degree of malignancy and recurrence.3.The expression level of xCT(SLC7A11)in U373TR cells was higher than that in U373 cells,and increased with the increase of temozolomide-resistant concentration in U373TR cells;the contents of cysteine and GSH in U87TR cells were higher than those in U87 cells.4.Silibinin inhibited the expression level of xCT(SLC7A11)in U87 and U87TR cells.5.Low-concentration silibinin combined with TMZ enhanced the killing effect of TMZ on U87 cells and inhibited the TMZ-induced increase in the expression level of xCT(SLC7A11).Conclusion:1.xCT(SLC7A11)is highly expressed in glioma tissue,and its expression increases with the increase of the malignancy of glioma tissue.2.High expression of xCT(SLC7A11)induces temozolomide resistance in glioma cells.3.Silibinin exerts anti-glioma cell viability and temozolomide resistance by inhibiting the expression of xCT(SLC7A11).4.Low-concentration silibinin combined with temozolomide reduces the temozolomide resistance of glioma cells and exerts a synergistic killing effect by inhibiting the expression of xCT(SLC7A11).更多还原