钙池操纵的钙内流在胶质瘤侵袭迁移中的作用

发布时间：2018-03-20 13:47

本文选题：胶质瘤　切入点：钙池操纵的钙内流　出处：《天津医科大学》2015年博士论文　论文类型：学位论文

【摘要】：研究目的:胶质瘤是中枢神经系统(central nervous system,CNS)最常见的肿瘤,其中胶质母细胞瘤(glioblastoma multiforme,GBM)恶性度最高,预后最差。胶质瘤浸润性生长的生物学特性使得手术不可能完全切除全部的肿瘤细胞,残存的肿瘤细胞对辅助放化疗的抵抗并继续在脑组织内侵袭迁移是胶质瘤复发及在CNS内播散的主要根源。钙离子作为细胞内第二信使,介导多种信号通路,参与调控细胞运动。最近研究发现,钙信号与恶性肿瘤的侵袭和转移有关。钙池操纵的钙内流(store-operated Ca2+entry,SOCE)由基质相互作用分子1(stromal interaction molecule 1,STIM1)与钙释放激活的钙通道蛋白1(Ca2+release-activated calcium channel protein 1,Orai1)介导,是非兴奋细胞内钙的来源途径之一。本研究拟探讨SOCE及其相关蛋白在胶质瘤侵袭迁移中的作用及机制,为胶质瘤的抗侵袭迁移治疗寻找新的靶点。研究方法:利用免疫组化、western blot检测人脑胶质瘤手术标本及胶质瘤细胞系中STIM1与Orai1的表达特征。为研究SOCE在胶质瘤细胞侵袭迁移过程中的作用,采用两种方式阻断SOCE:应用药理学抑制剂SKF96365干预或者RNA干扰敲低胶质瘤细胞中SOCE组成蛋白Orai1的表达;另外,为行恢复实验,在Orai1沉默的肿瘤细胞中重新表达目的蛋白。应用western blot、qRT-PCR验证Orai1敲低或重新表达的情况;应用Fluo-4/AM钙荧光指示剂法检测肿瘤细胞中SOCE的幅度变化;通过划痕实验、Transwell侵袭实验观察SOCE对胶质瘤细胞侵袭迁移能力的影响。通过黏着斑蛋白vinculin免疫荧光染色观察不同组别肿瘤细胞中黏着斑的位置、数目及大小;应用western blot检测E-cadherin、N-cadherin、vimentin的表达,观察SOCE对胶质瘤细胞中上皮间质转化(epithelial-to-mesenchymal transition,EMT)样改变的影响;应用western blot检测钙依赖性富含脯氨酸的酪氨酸激酶2(proline-rich tyrosine kinase 2,Pyk2)的总量及磷酸化的量,分析其活化程度。通过RNA干扰敲低Pyk2的表达,进一步研究SOCE是否是通过下游Pyk2信号通路发挥作用。应用SKF96365或者RNA干扰敲低C6胶质瘤细胞中Orai1的表达,研究SOCE对C6胶质瘤细胞体内外侵袭迁移能力的影响。研究结果:SOCE相关蛋白STIM1在非肿瘤性对照脑组织及各级别胶质瘤中均呈现低表达;而Orai1在对照脑组织中呈现低表达,在胶质瘤细胞系和胶质瘤标本中表达上调,且与肿瘤病理级别呈正相关。针对Orai1的RNA干扰能够不同程度抑制U251、SNB19胶质瘤细胞中SOCE的幅度。SKF96365及Orai1沉默能够抑制胶质瘤细胞的迁移及侵袭能力。与对照组相比,SKF96365处理组及Orai1沉默组细胞外周区域黏着斑的面积增大;同时,E-cadherin的表达升高,而N-cadherin、vimentin的表达降低。Pyk2活性分析提示SOCE受到抑制后,Pyk2总量不变而磷酸化Pyk2的表达降低。然而,Orai1重新表达均能够逆转Orai1沉默所引起的上述变化。Pyk2沉默能够抑制胶质瘤细胞的侵袭能力。Pyk2沉默组细胞外周区域黏着斑的面积增大;同时,E-cadherin的表达升高,而N-cadherin、vimentin的表达降低。SKF96365及Orai1沉默能够抑制C6细胞在体外的侵袭迁移能力。Orai1沉默能够抑制C6细胞在大鼠颅内的侵袭迁移,并抑制肿瘤细胞向血管旁聚集形成肿瘤小生境。结论:SOCE通过调节胶质瘤细胞中Pyk2的活性,继而调节细胞黏着斑周转的速度及EMT样改变,参与胶质瘤的侵袭迁移恶性生物学行为。下调SOCE能够抑制胶质瘤细胞在体内外的侵袭迁移,Orai1可能会成为抗胶质瘤侵袭治疗的新靶点。
[Abstract]:Objective: glioma is the central nervous system (central nervous system, CNS) the most common tumors, including glioblastoma (glioblastoma, multiforme, GBM) the most malignant glioma, the worst prognosis. The biological behavior of invasive growth makes surgery can not completely remove all of the tumor cells, chemotherapy and residual tumor the cell for resistance and continue in brain glioma recurrence and invasion is the main source of spread within CNS. Calcium as an intracellular second messenger mediated signaling pathways, is involved in the regulation of cell movement. A recent study found that calcium signaling and tumor invasion and metastasis of storeoperated. The calcium influx (store-operated Ca2+entry, SOCE) by stromal interaction molecule 1 (stromal interaction 1 molecule, STIM1) and calcium release activated calcium channel protein 1 (Ca2+release-activated calcium Channel protein 1, Orai1) mediated non excited intracellular calcium sources. This paper intends to discuss the SOCE and its associated protein in the invasion effect and mechanism of migration of glioma, to find new targets for anti glioma invasion and migration treatment. Methods: using immunohistochemistry, the expression characteristics of STIM1 Orai1 and Western blot detection of human glioma specimens and glioma cell lines. For the study of SOCE function in the process of invasion and migration of glioma cells, blocking the expression of SOCE: by pharmacological inhibitor SKF96365 intervention or RNA interference on SOCE glioma cells in low protein composition of Orai1 used in two ways; in addition, the recovery experiment for OK, re expression of target protein in Orai1 silencing in tumor cells. The application of Western blot qRT-PCR verification Orai1 knockdown or re expression of Fluo-4/AM; application of calcium fluorescent indicator was used to measure the tumor cells Variation of cell SOCE; through scratch test, Transwell invasion experiment the effects of SOCE on migration and invasion of glioma cells. Focal adhesions were observed in different groups of tumor cells in the position of focal adhesion protein vinculin staining by immunofluorescence, the number and size of blot; application of Western detection of E-cadherin, N-cadherin, vimentin expression, the effect of SOCE on the epithelial - mesenchymal transition in glioma cells (epithelial-to-mesenchymal transition, EMT) effects like change; tyrosine kinase by Western blot detection of calcium dependent proline rich 2 (proline-rich tyrosine 2 kinase, Pyk2) and the total amount of phosphorylated, analysis of its activation level. By RNA interference knockdown Pyk2 protein, further research whether SOCE play a role through Pyk2 signaling pathway. SKF96365 or RNA interference on Orai1 low expression in C6 glioma cells, S Effect of OCE on migration and invasion of C6 glioma cells in vitro and in vivo. Results: SOCE related protein STIM1 in non tumor control showed low expression of brain tissue and were all gliomas; while Orai1 showed low expression in normal brain tissue, expression in glioma cell lines and glioma specimens. And the pathological grade was positively correlated. In view of different degree of inhibition of U251 RNA interference of Orai1, SOCE in SNB19 glioma cells and the amplitude of.SKF96365 Orai1 silencing could inhibit the migration and invasion of glioma cells. Compared with the control group, SKF96365 group and Orai1 group of cells in the peripheral region of silent focal adhesion area increases at the same time, increased; the expression of E-cadherin and N-cadherin, decreased the expression of vimentin.Pyk2 activity analysis showed that SOCE inhibited the expression of Pyk2 after the same amount and phosphorylation of Pyk2 decreased. However, Orai1 re expression Can silence.Pyk2 these changes caused by the reversal of Orai1 silencing could inhibit glioma cell invasion ability of.Pyk2 cells in the peripheral region of silent group focal adhesion area increases; at the same time, increased the expression of E-cadherin and N-cadherin, vimentin and.SKF96365 decreased expression of Orai1 silencing can inhibit C6 cell migration in vitro invasion can silence.Orai1 inhibition of C6 cell invasion and migration in rat brain, and to inhibit the formation of tumor niche perivascular aggregation of tumor cells. Conclusion: SOCE glioma cells by regulating Pyk2 activity, and EMT like speed regulating cell and focal adhesion turnover changes in glioma invasion and migration of malignant biological behavior of invasion and migration of SOCE down. Can inhibit glioma cells in vitro and in vivo, Orai1 may become a new target for the treatment of invasive anti glioma.

【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R739.41

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