力、化学刺激对肝癌干细胞迁移、分化行为的影响及其相关分子机理

发布时间：2018-09-13 06:49

【摘要】：癌症是严重危害人类健康和生命的恶性疾病。在世界范围内,肿瘤死亡率前三位依次是肺癌、胃癌和肝癌。近年来肝癌的发病率不断上升,对肝癌的治疗和预防以及相关机理探索一直是肿瘤领域的研究热点。目前对于肝癌的治疗方案主要有外科手术切除、放射治疗、化学药物治疗以及肝移植等,但这些方法还不能从根本上解决其复发和转移问题,肝癌的预防和治疗仍然面临巨大的挑战。癌干细胞(cancer stem cell,CSCs)是恶性肿瘤组织中具有很强增殖能力、表现干细胞特性的细胞亚群,具备高度自我更新和分化潜能,不仅是恶性肿瘤的启动细胞,而且是形成不同分化程度癌细胞和癌症复发、转移及预后不良的根源,在癌症复发、转移中起决定作用,其靶向干预是癌症治疗的全新策略。癌症的发生发展是一个多因素调控的复杂过程,受到多种力学、化学因素的影响。在CSCs靶向干预的抗癌策略中,如何阻止CSCs转移或诱导CSCs分化都是CSCs靶向干预的重要手段,但目前人们对于力学、化学因素调控CSCs侵袭转移和分化等生物学行为的特征及相关机理还缺乏系统认识。为此,本课题以肝癌干细胞(liver cancer stem cells,LCSCs)为研究对象,首先考察了LCSCs的生物力学特质,然后研究了力学因素(剪切应力shear stress,SS)、化学因素(盐霉素salinomycin,Sal)对LCSCs转移、分化行为的影响及相关分子机制。本研究工作的主要内容和结果如下:(1)LCSCs的富集、鉴定及生物力学特质分析采用无血清干细胞专用培养液通过体外肿瘤球培养方法从高转移性肝癌细胞MHCC97H中筛选、富集LCSCs,对其癌干细胞特征及生物力学特质进行了分析。结果发现,筛选出的细胞相比母代MHCC97H细胞高表达癌干细胞标志物CD133、CD90、Oct3/4,具有更强的克隆形成能力、抗癌药物耐药性和裸鼠体内成瘤能力,表明筛选出的细胞具有癌干细胞表型,符合癌干细胞特征。Transwell迁移实验发现,LCSCs比MHCC97H细胞具有更强的迁移能力。原子力显微镜(atomic force microscopy,AFM)检测显示,LCSCs的杨氏模量显著小于MHCC97H细胞。激光共聚焦显微镜观察发现,LCSCs的细胞骨架F-actin呈点状结构,而MHCC97H细胞呈明显的丝状结构。Western blot实验进一步发现,与MHCC97H细胞相比,LCSCs中细胞骨架F-actin的表达显著减少。这些结果表明,LCSCs的生物力学特质与其高转移潜能有很密切的关系。(2)剪切应力通过FAK-ERK1/2-β-catenin信号通路促进LCSCs的迁移采用平行平板流动腔系统建立模拟体内lcscs血道转移模型,考察了血流剪切应力作用下lcscs迁移行为的变化及相关分子机制。实验结果发现,2dyne/cm2剪切应力加载6h后明显促进lcscs的迁移能力,粘着斑激酶(focaladhesionkinase,fak)和细胞外信号调节激酶1/2(extracellularsignalregulatedkinase1/2,erk1/2)磷酸化水平明显增加,β-链蛋白(β-catenin)的表达也明显增加,抑制fak或erk1/2激活或沉默β-catenin后,剪切应力促进的lcscs迁移受到明显抑制。免疫荧光染色观察发现,剪切应力加载后lcscsf-actin的表达下降,抑制fak、erk1/2后,也抑制了剪切应力诱导的f-actin下调。进一步利用afm检测了细胞杨氏模量的变化,结果发现剪切应力加载显著降低了lcscs的杨氏模量;抑制fak、erk1/2或β-catenin后,下降的lcscs杨氏模量得到了恢复。此外,fak抑制剂可阻断剪切应力诱导的erk1/2磷酸化以及β-catenin蛋白的表达,erk1/2抑制剂可阻断β-catenin蛋白的表达。上述结果提示,剪切应力可能通过fak-erk1/2-β-catenin信号途径使lcscs的细胞骨架f-actin重排,降低细胞硬度,从而增加lcscs的迁移能力。(3)剪切应力通过wnt/β-catenin信号通路调控lcscs的分化行为对lcscs施加2dyne/cm2剪切应力加载2天后,通过流式细胞术检测lcscs癌干细胞标志物cd133、cd90、oct3/4的表达。实验结果发现,剪切应力作用后cd133、cd90、oct3/4的表达显著下降。肿瘤球体形成实验发现,经2dyne/cm2剪切应力加载2天后,lcscs的球体形成能力显著下降;licl预处理激活wnt/β-catenin信号通路,剪切应力抑制的球体形成能力得到了恢复。药物敏感实验显示,2dyne/cm2剪切应力加载2天后,lcscs对顺铂(cisplatin)和5-氟尿嘧啶(5-fu)的敏感度增高,生存率明显下降。afm分析发现剪切应力加载后lcscs杨氏模量显著增加;利用licl激活β-catenin,抑制了剪切应力增加的细胞杨氏模量。westernblot检测发现,2dyne/cm2剪切应力作用lcscs2天后,β-catenin的表达明显下降,利用licl激活β-catenin,剪切应力促进的lcscs分化受到明显抑制,cd133、cd90、oct3/4的表达显著恢复。裸鼠体内致瘤实验表明,lcscs经剪切应力作用2天后,其致瘤能力明显下降,激活β-catenin,致瘤能力得到恢复。上述结果提示,2dyne/cm2剪切应力作用lcscs2天后可通过wnt/β-catenin信号通路使其发生分化。(4)盐霉素通过fak-erk1/2信号通路调控lcscs的运动能力transwell法检测发现,盐霉素明显抑制lcscs的迁移和侵袭能力,并在一定浓度范围内呈现浓度依赖性。westernblot检测表明,盐霉素作用lcscs后,fak和erk1/2磷酸化水平明显降低。此外,fak、erk1/2抑制剂也可抑制lcscs的迁移和侵袭能力。因此,盐霉素可能通过FAK-ERK1/2信号通路抑制LCSCs迁移和侵袭。明胶酶谱实验表明,盐霉素抑制LCSCs基质金属蛋白酶2(matrix metalloproteinase 2,MMP-2)和基质金属蛋白酶9(matrix metalloproteinase 9,MMP-9)分泌。此外,免疫荧光染色观察发现盐霉素作用后LCSCs F-actin的表达上升,FAK、ERK1/2抑制剂也可促进F-actin上调。进一步利用AFM检测发现盐霉素处理显著增加了LCSCs的杨氏模量,FAK、ERK1/2抑制剂作用后也升高了LCSCs的杨氏模量。上述结果提示,盐霉素可能通过FAK-ERK1/2信号通路促进F-actin表达,增加细胞硬度,减少MMP-2、MMP-9的分泌,从而抑制了LCSCs的运动能力。(5)盐霉素通过Wnt/β-catenin信号通路调控LCSCs的分化行为利用盐霉素对LCSCs处理2天后,通过流式细胞术检测发现LCSCs癌干细胞标志物CD133、CD90、Oct3/4的表达显著下降。肿瘤球体形成实验发现,经盐霉素处理后LCSCs的球体形成能力显著下降,利用LiCl激活β-catenin,肿瘤球体形成能力得到了恢复。药物敏感实验发现,盐霉素作用2天后的LCSCs,对于Cisplatin和5-FU的敏感度增高,生存率明显下降。此外,进一步利用AFM检测发现盐霉素可以显著增加LCSCs的杨氏模量。利用LiCl激活β-catenin后,细胞的杨氏模量得到明显恢复。Western blot检测表明,盐霉素作用LCSCs 2天后,β-catenin的表达明显下降,利用LiCl激活β-catenin,剪切应力促进的LCSCs分化受到明显抑制,LCSCs的癌干细胞标志物CD133、CD90、Oct3/4的表达显著恢复。裸鼠体内致瘤实验表明,LCSCs经盐霉素作用后,瘤体大小明显下降,激活β-catenin,致瘤能力得到恢复。上述结果提示,盐霉素作用LCSCs 2天后可通过Wnt/β-catenin信号通路使其发生了分化。综合上述结果,剪切应力在调控LCSCs转移、分化行为过程中起到了至关重要的作用,此外发现盐霉素可以抑制LCSCs转移、促使其分化。本研究结果为深入认识力、化学因素对LCSCs的作用机制提供了实验依据,为临床上通过靶向LCSCs治疗肝癌提供了理论指导。
[Abstract]:Cancer is a malignant disease that seriously endangers human health and life.In the world,the top three mortality rates of cancer are lung cancer,gastric cancer and liver cancer in turn.The incidence of liver cancer has been increasing in recent years.The treatment and prevention of liver cancer and the exploration of related mechanisms have been the focus of cancer research.At present,the treatment of liver cancer is the main program. Surgical resection, radiotherapy, chemotherapy and liver transplantation are needed, but these methods can not fundamentally solve the problem of recurrence and metastasis. The prevention and treatment of hepatocellular carcinoma still faces enormous challenges. Cell subsets, with a high degree of self-renewal and differentiation potential, are not only the initiator cells of malignant tumors, but also the source of formation of differentiated cancer cells and cancer recurrence, metastasis and poor prognosis. They play a decisive role in cancer recurrence and metastasis. Targeted intervention is a new strategy for cancer treatment. The complex process of multi-factor regulation is affected by many mechanical and chemical factors. In the anti-cancer strategy of CSCs targeted intervention, how to prevent CSCs from metastasis or induce CSCs differentiation is an important means of targeted intervention. However, at present, the characteristics and related mechanisms of biological behavior of CSCs are regulated by mechanical and chemical factors, such as invasion, metastasis and differentiation. In this study, the biomechanical properties of liver cancer stem cells (LCSCs) were investigated firstly, and then the effects of mechanical factors (shear stress, SS) and chemical factors (salinomycin, Sal) on the metastasis, differentiation and related molecular mechanisms of LCSCs were studied. The main contents and results of this study are as follows: (1) LCSCs enrichment, identification and biomechanical characteristics analysis. Serum-free stem cell culture medium was used to screen and enrich LCSCs from high metastatic hepatocellular carcinoma cell line MHCC97H in vitro. The characteristics and biomechanical characteristics of LCSCs were analyzed. Transwell migration assay showed that LCSCs were finer than MHCC97H cells in the morphology of cancer stem cells. Atomic force microscopy (AFM) showed that the Young's modulus of LCSCs was significantly lower than that of MHCC97H cells. Confocal laser microscopy showed that the F-actin cytoskeleton of LCSCs was dotted, while MHCC97H cells showed obvious filamentous structure. These results indicate that the biomechanical properties of LCSCs are closely related to their high metastatic potential. (2) Shear stress promotes the migration of LCSCs through FAK-ERK1/2-beta-catenin signaling pathway. Parallel plate flow chamber system is used to establish blood channel metastasis model of LCSCs in vivo. The results showed that 2dyne/cm2 shear stress could significantly promote the migration of lcscs, focal adhesion kinase (fak) and extracellular signal regulated kinase 1/2 (erk1/2) phosphoric acid. Immunofluorescence staining showed that the expression of lcscsf-actin decreased after shear stress loading, and the expression of FAK and erk1/2 was inhibited after inhibiting the activation of FAK or erk1/2 or silencing of beta-catenin. Furthermore, AFM was used to detect the changes of young's modulus of lcscs. the results showed that shear stress loading significantly reduced the young's modulus of lcscs. after inhibiting fak, ERK1 / 2 or beta-catenin, the decreased young's modulus of LCSCs was restored. furthermore, FAK inhibitor could block shear stress-induced ERK1 / 2 phosphorylation and beta-catenin protein. These results suggest that shear stress may regulate the differentiation of LCSCs through the fak-erk1/2-beta-catenin signaling pathway through the fak-erk1/2-beta-catenin signaling pathway by rearranging the cytoskeleton F-actin and reducing the cell hardness. The expression of cd133, CD90 and OCT3 / 4 was detected by flow cytometry after loading LCSCs with 2dyne / cm2 shear stress for 2 days. the results showed that the expression of cd133, CD90 and OCT3 / 4 was significantly decreased after loading LCSCs with 2dyne / cm2 shear stress. LiCl pretreatment activated the Wnt / beta-catenin signaling pathway and restored the ability of sphere formation inhibited by shear stress. Young's modulus of LCSCs increased significantly after loading, and was inhibited by activation of beta-catenin with licl. Western blot analysis showed that the expression of beta-catenin decreased significantly after 2 days of shear stress, and the differentiation of LCSCs promoted by shear stress was inhibited by activation of beta-catenin with licl, cd133, cd90, oc. The expression of t3/4 was significantly restored in nude mice. The tumorigenic ability of LCSCs decreased significantly after 2 days of shear stress, and the tumorigenic ability of LCSCs was restored by activating beta-catenin. These results suggest that 2dyne/cm2 shear stress can differentiate LCSCs through wnt/beta-catenin signaling pathway after 2 days of shear stress. (4) Salinomycin can differentiate LCSCs through fak-erk1 signaling pathway. Transwell assay showed that salinomycin significantly inhibited the migration and invasion of LCSCs in a concentration-dependent manner. Western blot assay showed that the phosphorylation levels of FAK and ERK1 / 2 were significantly decreased after salinomycin acted on lcscs. Therefore, salinomycin may inhibit the migration and invasion of LCSCs through FAK-ERK1/2 signaling pathway. Gelatin zymographic experiments showed that salinomycin inhibited the secretion of matrix metalloproteinase 2 (MMP-2) and matrix metalloproteinase 9 (MMP-9) in LCSCs. In addition, immunofluorescence staining was used to observe the effect of salinomycin on the secretion of matrix metalloproteinase 9 (MMP-9). It was found that the expression of F-actin in LCSCs increased after salinomycin treatment, and that FAK and ERK1/2 inhibitors could also promote the up-regulation of F-actin. Further, it was found that salinomycin treatment significantly increased the Young's modulus of LCSCs, FAK and ERK1/2 inhibitors also increased the Young's modulus of LCSCs. These results suggest that salinomycin may be through FAK-ERK1/2 signal. (5) Salinomycin regulates the differentiation of LCSCs by Wnt/beta-catenin signaling pathway. After two days of treatment with salinomycin, the expression of CD133, CD90 and Oct3/4 in LCSCs was detected by flow cytometry. Tumor spherogenesis test showed that the spherogenesis ability of LCSCs decreased significantly after salinomycin treatment, and the ability of tumor spherogenesis was restored by activating beta-catenin with LiCl. Drug sensitivity test showed that the sensitivity of LCSCs to Cisplatin and 5-FU increased and the survival rate decreased significantly after salinomycin treatment for 2 days. Furthermore, the Young's modulus of LCSCs was significantly increased by salinomycin. After activation of beta-catenin by LiCl, the Young's modulus of LCSCs was significantly restored. Western blot analysis showed that the expression of beta-catenin was significantly decreased after 2 days of salinomycin treatment, and the differentiation of LCSCs was promoted by shear stress after activation of beta-catenin by LiCl. The expression of CD133, CD90 and Oct3/4 in LCSCs was significantly restored. The tumorigenic experiments in nude mice showed that the size of LCSCs decreased significantly after salinomycin treatment, and the tumorigenicity was restored by activating beta-catenin. These results suggest that salinomycin can induce LCSCs to develop through Wnt/beta-catenin signaling pathway after 2 days. In conclusion, shear stress plays an important role in regulating the metastasis and differentiation of LCSCs. Salinomycin can inhibit the metastasis and promote the differentiation of LCSCs. The results of this study provide experimental evidence for further understanding of the mechanism of chemical factors on LCSCs and for clinical targeting of LCSCs. It provides theoretical guidance for the treatment of liver cancer.
【学位授予单位】：重庆大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.7

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