【作者】 张涛；

【导师】 王志平；

【作者基本信息】 兰州大学 ， 临床医学·外科学， 2019， 硕士

【摘要】 目的:全球范围内,每年有将近150000人死于膀胱癌。目前,针对进展期膀胱,主要有内腔镜和开放手术、局部或系统性免疫治疗、放化疗等治疗方案,尽管这些治疗策略已经改善了治疗效果并延长了膀胱癌患者的生存期以及生活治疗,但该疾病仍然无法完全治愈而且还要具有高复发风险,并治疗预后均不太理想。近些年来学者们发现,有一群未或低分化的膀胱肿瘤细胞,他们称之为膀胱肿瘤干细胞,他们认为肿瘤的复发与不良预后与膀胱肿瘤干细胞有关。现如今已有许多关于膀胱肿瘤干细胞分子标志物的研究被发表,CD44、Ck5、CD47、ALDH等分子被认为是肿瘤干细胞的分子标志物,科学家们基于这些分子标志物的研究,为膀胱癌的治疗提供新的研究方向与方法。将旧药重新用于新用途,例如在针对肿瘤的临床治疗中使用旧药去达到抑癌效果是一个非常有吸引力的,令人兴奋的领域。双硫仑就是类似的一个很好的例子。双硫仑由于其独特的已知特性,低成本,低副作用和对不同癌症的高选择性以及与其他药物的协同活性而被重新用作潜在的抗癌药物。而且双硫仑抑制肿瘤细胞的机制之一就是作为ALDH的抑制剂。本次实验探讨双硫仑对膀胱肿瘤中ALDH1A1表达的影响及其增强肿瘤细胞对顺铂敏感性的潜在机制。方法:将实验分为对照组和双硫仑(DSF)组。检索相关数据库,查询ALDH1A1膀胱癌标本中的表达情况。提取EJ细胞与SV-HUC-1细胞的mRNA,检测两者ALDH1A1的表达情况。采用克隆形成实验检测DSF对膀胱肿瘤细胞成团能力的影响。提取DSF干预组与对照组膀胱肿瘤细胞RNA,采用实时荧光定量PCR检测DSF处理后,肿瘤细胞内ALDH1A1及其他干性相关基因的表达差异。通过划痕实验检测DSF干预后0H以及24H时,DSF对膀胱肿瘤细胞迁移能力的影响。流式周期检测DSF干预后对于膀胱肿瘤细胞周期的影响。利用CCK8法以及流式检测细胞凋亡方法检测DSF对肿瘤细胞顺铂敏感性的影响。结果:DSF组膀胱肿瘤细胞的克隆成球数目明显低于对照组。对照组和DSF组克隆形成后,用结晶紫将球团染色,溶解结晶紫后,两组溶液570nm的吸光度分别为0.884±0.022和0.490±0.027,差异有明显统计学意义(P<0.001)。与正常尿路上皮细胞SV-HUC-1相比,膀胱癌EJ细胞中ALDH1A1的表达量明显增高,差异具有统计学意义(P<0.001)。与对照组相比,DSF组中细胞的ALDH1A1的表达水平明显下调,NOTCH2,SHH等干细胞基因的表达亦有下调,差异均有统计学意义(P<0.05)。与对照组相比,DSF干预后的膀胱肿瘤细胞的迁移能力明显下降。与对照组相比,DSF干预后的膀胱肿瘤细胞处于G1期的数量明显增加,而S期细胞数量下降。顺铂联合DSF与单用顺铂两种方法分别对膀胱肿瘤细胞干预24h,测得顺铂的中位杀细胞浓度分别为8umol和32umol,差异亦具有统计学意义(P<0.05),说明在DSF干预后的膀胱肿瘤细胞对顺铂变得敏感。结论:膀胱癌组织较癌旁组织有较高的ALDH1A1表达。膀胱癌细胞ALDH1A1表达高于正常尿路上皮细胞。双硫仑作为ALDH的抑制剂,可通过降低膀胱肿瘤细胞内ALDH1A1的表达,影响膀胱肿瘤细胞的迁移、克隆形成能力以及影响细胞周期等表型,并且增强了膀胱肿瘤细胞对于顺铂的敏感性。更多还原

【Abstract】 Objective: Worldwide,nearly 150,000 people die of bladder cancer each year.At present,for advanced bladder,there are mainly endoscopic and open surgery,local or systemic immunotherapy,radiotherapy and chemotherapy,etc.,although these treatment strategies have improved the treatment effect and prolonged the survival and life treatment of bladder cancer patients.However,the disease is still not completely cured and has a high risk of recurrence,and the prognosis is not ideal.In recent years,scholars have found that there are a group of undifferentiated bladder tumor cells,which are called bladder cancer stem cells.They believe that tumor recurrence and poor prognosis are related to bladder cancer stem cells.Many studies on bladder cancer stem cells have been published,and CD44,Ck5,CD47,ALDH and other molecules are considered to be molecular markers of cancer stem cells.Based on these molecular markers of bladder cancer stem cells,new treatments for bladder cancer are provided.Ideas and methods.Reusing old medicines for new purposes,such as using old medicines to achieve cancer suppression in clinical treatment of tumors,is a very attractive and exciting area.Disulfiram is a good example of this.Disulfiram is re-used as a potential anticancer drug due to its unique known properties,low cost,low side effects and high selectivity for different cancers and synergistic activity with other drugs.Moreover,one of the mechanisms by which disulfiram inhibits tumor cells is as an inhibitor of ALDH.This experiment explored the effect of disulfiram on the expression of ALDH1A1 in bladder tumors and its potential mechanism of enhancing the sensitivity of tumor cells to cisplatin.Methods: The experiment was divided into a control group and a disulfiram(DSF)group.Search the relevant database to query the expression of ALDH1A1 bladder cancer specimens.The mRNA of EJ cells and SV-HUC-1 cells were extracted,and the expression of ALDH1A1 was detected.The effect of DSF on the colonization ability of bladder tumor cells was examined by colony formation assay.The RNA of bladder tumor cells in DSF intervention group and control group were extracted,and the expression of ALDH1A1 and other dry related genes in tumor cells were detected by real-time fluorescent quantitative PCR after real-time quantitative PCR.The effect of DSF on the migration ability of bladder tumor cells was examined by scratch test at 0H and 24 H after DSF intervention.The effect of flow cycle on the cell cycle of bladder tumors after DSF intervention.The effect of DSF on the sensitivity of tumor cells to cisplatin was detected by CCK8 method and flow cytometry.Results: The number of cloned cells in the bladder cancer cells of the DSF group was significantly lower than that of the control group.After the clones of the control group and the DSF group were formed,the pellets were stained with crystal violet.After dissolving the crystal violet,the absorbance at 570 nm of the two groups was 0.884±0.022 and 0.490±0.027,respectively,and the difference was statistically significant(P<0.001).Compared with normal urothelial cells SV-HUC-1,the expression of ALDH1A1 in bladder cancer EJ cells was significantly increased,and the difference was statistically significant(P<0.001).Compared with the control group,the expression level of ALDH1A1 in DSF group was significantly down-regulated.The expression of stem cell genes such as NOTCH2 and SHH was also down-regulated,and the difference was statistically significant(P<0.05).Compared with the control group,the migration ability of bladder tumor cells after DSF intervention was significantly decreased.Compared with the control group,the number of bladder tumor cells in the G1 phase increased significantly after DSF intervention,while the number of cells in the S phase decreased.The cisplatin combined with DSF and cisplatin alone were used to treat bladder tumor cells for 24 h.The median cell killing concentrations of cisplatin were 8umol and 32 umol,respectively,and the difference was statistically significant(P<0.05).Bladder tumor cells after DSF intervention become sensitive to cisplatin.Conclusion: Bladder cancer tissue has higher ALDH1A1 expression than adjacent tissues.The expression of ALDH1A1 in bladder cancer cells is higher than that in normal urothelial cells.As an inhibitor of ALDH,disulfiram can affect the expression of ALDH1A1 in bladder tumor cells,affecting the migration,clone formation ability and cell cycle phenotype of bladder tumor cells,it also enhances the sensitivity of bladder tumor cells to cisplatin.更多还原