本文選題：膠質(zhì)瘤　切入點：鈣池操縱的鈣內(nèi)流　出處：《天津醫(yī)科大學》2015年博士論文　論文類型：學位論文

【摘要】：研究目的:膠質(zhì)瘤是中樞神經(jīng)系統(tǒng)(central nervous system,CNS)最常見的腫瘤,其中膠質(zhì)母細胞瘤(glioblastoma multiforme,GBM)惡性度最高,預后最差。膠質(zhì)瘤浸潤性生長的生物學特性使得手術(shù)不可能完全切除全部的腫瘤細胞,殘存的腫瘤細胞對輔助放化療的抵抗并繼續(xù)在腦組織內(nèi)侵襲遷移是膠質(zhì)瘤復發(fā)及在CNS內(nèi)播散的主要根源。鈣離子作為細胞內(nèi)第二信使,介導多種信號通路,參與調(diào)控細胞運動。最近研究發(fā)現(xiàn),鈣信號與惡性腫瘤的侵襲和轉(zhuǎn)移有關。鈣池操縱的鈣內(nèi)流(store-operated Ca2+entry,SOCE)由基質(zhì)相互作用分子1(stromal interaction molecule 1,STIM1)與鈣釋放激活的鈣通道蛋白1(Ca2+release-activated calcium channel protein 1,Orai1)介導,是非興奮細胞內(nèi)鈣的來源途徑之一。本研究擬探討SOCE及其相關蛋白在膠質(zhì)瘤侵襲遷移中的作用及機制,為膠質(zhì)瘤的抗侵襲遷移治療尋找新的靶點。研究方法:利用免疫組化、western blot檢測人腦膠質(zhì)瘤手術(shù)標本及膠質(zhì)瘤細胞系中STIM1與Orai1的表達特征。為研究SOCE在膠質(zhì)瘤細胞侵襲遷移過程中的作用,采用兩種方式阻斷SOCE:應用藥理學抑制劑SKF96365干預或者RNA干擾敲低膠質(zhì)瘤細胞中SOCE組成蛋白Orai1的表達;另外,為行恢復實驗,在Orai1沉默的腫瘤細胞中重新表達目的蛋白。應用western blot、qRT-PCR驗證Orai1敲低或重新表達的情況;應用Fluo-4/AM鈣熒光指示劑法檢測腫瘤細胞中SOCE的幅度變化;通過劃痕實驗、Transwell侵襲實驗觀察SOCE對膠質(zhì)瘤細胞侵襲遷移能力的影響。通過黏著斑蛋白vinculin免疫熒光染色觀察不同組別腫瘤細胞中黏著斑的位置、數(shù)目及大小;應用western blot檢測E-cadherin、N-cadherin、vimentin的表達,觀察SOCE對膠質(zhì)瘤細胞中上皮間質(zhì)轉(zhuǎn)化(epithelial-to-mesenchymal transition,EMT)樣改變的影響;應用western blot檢測鈣依賴性富含脯氨酸的酪氨酸激酶2(proline-rich tyrosine kinase 2,Pyk2)的總量及磷酸化的量,分析其活化程度。通過RNA干擾敲低Pyk2的表達,進一步研究SOCE是否是通過下游Pyk2信號通路發(fā)揮作用。應用SKF96365或者RNA干擾敲低C6膠質(zhì)瘤細胞中Orai1的表達,研究SOCE對C6膠質(zhì)瘤細胞體內(nèi)外侵襲遷移能力的影響。研究結(jié)果:SOCE相關蛋白STIM1在非腫瘤性對照腦組織及各級別膠質(zhì)瘤中均呈現(xiàn)低表達;而Orai1在對照腦組織中呈現(xiàn)低表達,在膠質(zhì)瘤細胞系和膠質(zhì)瘤標本中表達上調(diào),且與腫瘤病理級別呈正相關。針對Orai1的RNA干擾能夠不同程度抑制U251、SNB19膠質(zhì)瘤細胞中SOCE的幅度。SKF96365及Orai1沉默能夠抑制膠質(zhì)瘤細胞的遷移及侵襲能力。與對照組相比,SKF96365處理組及Orai1沉默組細胞外周區(qū)域黏著斑的面積增大;同時,E-cadherin的表達升高,而N-cadherin、vimentin的表達降低。Pyk2活性分析提示SOCE受到抑制后,Pyk2總量不變而磷酸化Pyk2的表達降低。然而,Orai1重新表達均能夠逆轉(zhuǎn)Orai1沉默所引起的上述變化。Pyk2沉默能夠抑制膠質(zhì)瘤細胞的侵襲能力。Pyk2沉默組細胞外周區(qū)域黏著斑的面積增大;同時,E-cadherin的表達升高,而N-cadherin、vimentin的表達降低。SKF96365及Orai1沉默能夠抑制C6細胞在體外的侵襲遷移能力。Orai1沉默能夠抑制C6細胞在大鼠顱內(nèi)的侵襲遷移,并抑制腫瘤細胞向血管旁聚集形成腫瘤小生境。結(jié)論:SOCE通過調(diào)節(jié)膠質(zhì)瘤細胞中Pyk2的活性,繼而調(diào)節(jié)細胞黏著斑周轉(zhuǎn)的速度及EMT樣改變,參與膠質(zhì)瘤的侵襲遷移惡性生物學行為。下調(diào)SOCE能夠抑制膠質(zhì)瘤細胞在體內(nèi)外的侵襲遷移,Orai1可能會成為抗膠質(zhì)瘤侵襲治療的新靶點。
[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.

【學位授予單位】：天津醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R739.41

【參考文獻】

相關期刊論文 前2條

1 王麗娟;趙紅;陳文媛;耿越;;鈣庫調(diào)控鈣內(nèi)流信號機制的研究進展[J];科技信息;2009年27期

2 中國腦膠質(zhì)瘤協(xié)作組;王偉民;白紅民;施沖;何洹;楊學軍;王任直;馬文斌;周良輔;吳勁松;江濤;李少武;王引言;陳新忠;;喚醒狀態(tài)下切除腦功能區(qū)膠質(zhì)瘤手術(shù)技術(shù)的專家共識[J];中國微侵襲神經(jīng)外科雜志;2013年03期

，

本文編號：1639342