發(fā)布時間：2018-05-22 12:35

  本文選題：膀胱癌 + 鞘氨醇激酶1　； 參考：《第三軍醫(yī)大學(xué)》2015年博士論文

【摘要】：背景膀胱癌(Bladder cancer,BC)是泌尿系統(tǒng)中易發(fā)的首位腫瘤,具有較高的發(fā)病率和致死率。目前治療措施主要有腔內(nèi)膀胱腫瘤切除,根治性膀胱切除等手術(shù)、適形放療和局部灌注或全身化療等,但是經(jīng)上述種種措施單獨(dú)或合并治療后復(fù)查時還經(jīng)常出現(xiàn)腫瘤再發(fā)甚至轉(zhuǎn)移,預(yù)后不良。臨床數(shù)據(jù)分析顯示自2007年至2012年共有來自我們國家44個大型泌尿外科中心共14260例膀胱癌患者,其中約70%以上的患者接收治療后出現(xiàn)再發(fā)轉(zhuǎn)移擴(kuò)散,30%的再發(fā)腫瘤惡性度較前增加。隨著分子靶向治療在腫瘤綜合治療中逐漸成為臨床熱點(diǎn),膀胱癌中有無合適的分子靶點(diǎn)以及如何利用該靶點(diǎn)來減緩腫瘤的再發(fā)和進(jìn)展將具有肯定的現(xiàn)實性意義。脂類組學(xué)的研究發(fā)現(xiàn)鞘脂也可以具有信號調(diào)控的作用。如神經(jīng)酰胺(Ceramide,Cer)、鞘氨醇(Sphingosine,Sph),可以調(diào)控細(xì)胞內(nèi)信號通路引起細(xì)胞周期阻滯和促進(jìn)細(xì)胞凋亡;鞘氨醇1-磷酸(Sphingosine 1-phosphate,S1P)則有促進(jìn)細(xì)胞增殖的功能。Cer/Sph與S1P在細(xì)胞中的水平存在動態(tài)平衡,而它們的作用一種促死與一種促生,正好相反,水平的平衡決定了作用的平衡,作用的平衡決定了該細(xì)胞的命運(yùn)是活或死。這種現(xiàn)象就是所謂的“鞘脂可調(diào)變阻器”(Sphingolipid-rheostat function)。研究認(rèn)為鞘氨醇激酶族(Sphingosine kinases,SphKs)是調(diào)控這個平衡的最重要酶族,它能將促死的鞘氨醇、神經(jīng)酰胺轉(zhuǎn)化為促生的磷酸鞘氨醇。通過抑制此族蛋白的活性則能將可調(diào)變阻器調(diào)向細(xì)胞的死亡方向起到抗腫瘤的作用。發(fā)現(xiàn)Sph K1可作為治療腫瘤的手段。腫瘤細(xì)胞侵襲與轉(zhuǎn)移是影響腫瘤患者預(yù)后的重要原因。膀胱尿路上皮癌細(xì)胞的轉(zhuǎn)移需要?dú)v經(jīng)癌細(xì)胞-基底細(xì)胞黏附、細(xì)胞-基質(zhì)黏附的破壞,細(xì)胞外介質(zhì)蛋白的水解,以及新生血管生成的一系列過程,抗腫瘤研究就是要研究如何阻斷這一系列過程,進(jìn)而阻斷癌細(xì)胞的轉(zhuǎn)移,侵襲及擴(kuò)散,同樣是膀胱癌防治工作的重點(diǎn)。我們已知癌細(xì)胞侵襲、遷移的過程中,基質(zhì)金屬蛋白酶家族(Matrix-metalloproteinase,MMPs)的降解既是腫瘤侵襲與癌細(xì)胞遷移中組織重構(gòu)所必需的,也是腫瘤侵襲早期的變化之一。惡變細(xì)胞通過游離的MMPs發(fā)揮降解細(xì)胞外基質(zhì)、促新生血管形成的雙重作用而利于其侵襲。基質(zhì)金屬蛋白酶家族中的成員MMP-2與MMP-9能夠降解Ⅳ型膠原蛋白進(jìn)而促進(jìn)基質(zhì)膜的降解。在多種腫瘤細(xì)胞的侵襲與轉(zhuǎn)移過程中它們的表達(dá)都出現(xiàn)升高,兩種現(xiàn)象具有相關(guān)性。細(xì)胞外反應(yīng)激酶(extracellularresponsekinases,erk)是mapk蛋白家族中的一種蛋白,有酶活性,其生物學(xué)作用是能夠控制細(xì)胞增殖和分化、調(diào)控細(xì)胞凋亡、決定細(xì)胞惡性變,并且能夠維持細(xì)胞形態(tài)。erk的表達(dá)升高及激活狀態(tài)上調(diào)可以在許多的人類惡性腫瘤中檢測到。p-erk1/2是erk1/2的活化形式,通過erk1/2的202位l-蘇氨酸(l-threonine,thr202)與204位酪氨酸(tyrosine,tyr204)出現(xiàn)磷酸化具有活性,作用較erk1/2增強(qiáng)。文獻(xiàn)復(fù)習(xí)發(fā)現(xiàn)在許多腫瘤中sphk1/s1p激活erk1/2通路從而促進(jìn)癌細(xì)胞侵襲與遷移。尿激酶型纖維蛋白溶酶原激活物(urokinase-typeplasminogenactivator,upa)能夠?qū)⒀獫{纖維蛋白溶酶原活化為酶并能夠獨(dú)立地或者通過活化的酶降解細(xì)胞外基質(zhì)類物質(zhì)、激活mmps、引起促腫瘤因子以及vegf的釋放促進(jìn)細(xì)胞侵襲性增強(qiáng),并能加速腫瘤細(xì)胞的遷移能力。既往研究發(fā)現(xiàn)通過抑制erk1/2通路,經(jīng)過一系列信號轉(zhuǎn)導(dǎo)、放大、反饋等方式,可出現(xiàn)mmp-2/9、upa表達(dá)的下調(diào),它們協(xié)同起來可以實現(xiàn)抑制腫瘤轉(zhuǎn)移的目的。本研究即以sphk1作為對象,明確sphk1在bc患者中表達(dá)是否存在異常,其水平與臨床病理特點(diǎn)及疾病預(yù)后是否關(guān)聯(lián),并將探討其細(xì)胞水平及分子水平的機(jī)制。具體方式為應(yīng)用調(diào)控sphk1蛋白表達(dá)的化學(xué)藥物作用于膀胱癌腫瘤細(xì)胞biu-87,觀察膀胱腫瘤細(xì)胞biu-87被藥物處理后的侵襲和遷移能力的變化,并測定細(xì)胞內(nèi)erk1/2與perk1/2蛋白,mmp-2、mmp-9、upa及vegf蛋白分泌的變化,從細(xì)胞及分子水平明確鞘氨醇激酶1有無改變?nèi)税螂装┘?xì)胞的侵襲和遷移能力的作用,其作用是增強(qiáng)還是減弱,進(jìn)而與臨床病理結(jié)果相對應(yīng),為以鞘氨醇激酶1為靶點(diǎn)的分子腫瘤治療學(xué)初步言明機(jī)制。研究目的探討sphk1在膀胱尿路上皮癌發(fā)生發(fā)展中的作用及其機(jī)制,為將sphk1作為膀胱尿路上皮癌的診斷治療之分子靶點(diǎn)提供理論依據(jù)。材料、方法和結(jié)果以細(xì)胞及其中分子為研究對象用佛波醇12-肉豆蔻酸酯13-乙酸酯(phorbol12-myristate13-acetate,pma)為sphk1激活劑;二甲基鞘氨醇(n-dimethyld-erythrosphingsine,dms)為sphk1抑制劑;0.9%nacl為空白試劑將膀胱癌biu-87細(xì)胞株分別處理為激活組、抑制組和空白對照組。cck-8法測各組細(xì)胞的增殖情況;Transwell小室模型測定各組BIU-87細(xì)胞的相對侵襲率與遷移率變化;Western-blot法測各組細(xì)胞Sphk1、ERK1/2與p-ERK1/2蛋白水平;ELISA方法檢測各組細(xì)胞上清中MMP-2、MMP-9、VEGF及uPA的蛋白含量的表達(dá);qRT-PCR測各組細(xì)胞Sph K1、MMP-2、MMP-9、uPA的mRNA水平;Matrigel三維培養(yǎng)法觀察各組細(xì)胞血管生成擬態(tài)(vasculogenic mimicry,VM)形成能力。結(jié)果:SphK1激活劑可促進(jìn)BIU-87細(xì)胞侵襲與遷移,同時明顯增強(qiáng)細(xì)胞Sph K1、ERK1/2與p-ERK1/2的蛋白表達(dá),并促進(jìn)MMP-2、MMP-9及u PA的蛋白分泌。促進(jìn)Matrigel構(gòu)建的三維培養(yǎng)系統(tǒng)中VM的形成;明顯增強(qiáng)VEGF蛋白表達(dá)。Sph K1抑制劑則能抑制BIU-87細(xì)胞侵襲與遷移,同時抑制Sph K1、ERK1/2與p-ERK1/2的蛋白表達(dá),并抑制MMP-2、MMP-9及u PA的蛋白分泌;三維培養(yǎng)中不能形成管狀VM;明顯減弱VEGF蛋白表達(dá)。以患者臨床資料為研究對象通過q RT-PCR和IHC方法檢測患者膀胱癌組織和鄰近的正常組織Sphk1的mRNA和蛋白質(zhì)的表達(dá),研究其差異,并分析其與膀胱癌臨床病理特征的關(guān)系。結(jié)果:在37對膀胱癌組織及臨近正常組織中測量發(fā)現(xiàn)癌組織中SphK1m RNA的表達(dá)顯著高于癌旁正常組織中的表達(dá);并發(fā)現(xiàn)153例膀胱癌病理組織切片中Sph K1蛋白表達(dá)與膀胱癌病理分期(P=0.045)、腫瘤病理分級(P0.001)顯著相關(guān)。通過繪制Kaplan-Meier生存曲線發(fā)現(xiàn)高表達(dá)Sph K1患者的5年生存率顯著下降(P0.001)。多變量Cox回歸分析顯示SphK1的高表達(dá)可以作為這種疾病的一種獨(dú)立的不良預(yù)后因素。結(jié)論Sph K1在膀胱尿路上皮癌發(fā)生發(fā)展中發(fā)揮著重要的作用,其機(jī)制可能與激活信號ERK1/2通路從而促進(jìn)MMP-2、MMP-9及u PA蛋白分泌有關(guān),進(jìn)而促進(jìn)VEGF表達(dá)并增強(qiáng)腫瘤細(xì)胞的侵襲遷移能力而發(fā)揮作用。我們可以將Sph K1作為膀胱尿路上皮癌的診斷治療之分子靶點(diǎn)。
[Abstract]:Bladder cancer (BC) is the most likely primary tumor in the urinary system, with high morbidity and mortality. The main treatment measures are intracavitary bladder tumor resection, radical cystectomy, conformal radiotherapy, local perfusion or systemic chemotherapy, but these measures are reviewed alone or after combined treatment. The clinical data analysis showed that from 2007 to 2012, there were 14260 cases of bladder cancer from 44 large department of Urology centers in our country, of which more than 70% of the patients received recurrent metastasis after receiving treatment, and 30% of the recurrent tumors were more malignant than before. Targeted therapy has gradually become a clinical hotspot in the comprehensive treatment of tumor. It is of certain practical significance whether the bladder cancer has a suitable molecular target and how to use the target to slow down the recurrence and progression of the tumor. Sphingosine (Sph) can regulate the intracellular signaling pathway to cause cell cycle arrest and promote cell apoptosis; Sphingosine 1-phosphate (S1P) has a functional.Cer/Sph that promotes cell proliferation and has a dynamic balance in the level of S1P in the cell, and their role is to promote death and a kind of growth promoting, just the opposite, water. Balance determines the balance of action. The balance of the action determines whether the cell's fate is alive or dead. This phenomenon is called "Sphingolipid-rheostat function". It is considered that the Sphingosine kinases (SphKs) is the most important group of enzymes that regulate this balance, and it can promote the sheath of death. Ammonia alcohol, ceramide is converted to sphingosine phosphate. By inhibiting the activity of this protein, an adjustable rheostat can play an antitumor role in the direction of cell death. It is found that Sph K1 can be used as a means of cancer treatment. Tumor cell invasion and metastasis are important factors affecting the prognosis of tumor patients. Cell metastasis needs to undergo cancer cell adhesion to basal cells, destruction of cell matrix adhesion, hydrolysis of extracellular matrix protein, and a series of processes of angiogenesis. Antitumor research is to study how to block this series of processes, and then block the transfer, invasion and diffusion of cancer cells, which are also the prevention and treatment of bladder cancer. In the process of cancer cell invasion and migration, the degradation of Matrix-metalloproteinase (MMPs) is not only necessary for tumor invasion and tissue remodeling in cancer cell migration, but also one of the changes in the early stage of tumor invasion. Malignant cells degrade the extracellular matrix through free MMPs and promote the neovascularization. The members of the matrix metalloproteinase family, MMP-2 and MMP-9, can degrade type IV collagen and then promote the degradation of the matrix membrane. In the invasion and metastasis of a variety of tumor cells, their expressions are elevated, and the two phenomena are related. The extracellular reaction kinase (extracellularresp Onsekinases, ERK) is a protein in the MAPK protein family, which has an enzyme activity. Its biological function is to control cell proliferation and differentiation, regulate cell apoptosis, determine cell malignant transformation, and maintain the expression of.Erk in cell morphology and the up-regulated activation state, which can detect.P-erk1/2 as erk1/2 in many human malignant tumors. The activation form is activated by phosphorylation of 202 - bit l- threonine (l-threonine, thr202) with 204 - bit tyrosine (tyrosine, tyr204) in erk1/2. The effect is stronger than erk1/2. The literature review found that sphk1/s1p activates the erk1/2 pathway in many tumors to promote cancer cell invasion and migration. The urokinase type fibrinolytic activator (uro) Kinase-typeplasminogenactivator, uPA) can activate plasma fibrinolytic enzyme into enzymes and can degrade extracellular matrix substances independently or through activated enzymes, activate MMPs, cause the release of tumor stimulating factors and VEGF to promote cell invasiveness and increase the migration ability of tumor cells. The erk1/2 pathway, through a series of signal transduction, amplification, and feedback, can reduce the expression of mmp-2/9 and uPA, and they cooperate to suppress tumor metastasis. This study is based on SphK1 as an object to determine whether the expression of SphK1 in BC patients is abnormal, its level and clinicopathological characteristics and the prognosis of the disease are No correlation, and the mechanism of cell level and molecular level. The specific way is to use the chemical drugs that regulate the expression of SphK1 protein in bladder cancer cell BIU-87, to observe the invasion and migration of BIU-87 in bladder tumor cells after treatment, and to determine the erk1/2 and perk1/2 protein, MMP-2, MMP-9, uPA and uPA in cell. The changes in the secretion of VEGF protein, from the cell and molecular level to determine whether sphingosine kinase 1 has the effect of changing the invasion and migration of human bladder cancer cells, its effect is enhanced or weakened, and corresponded to the clinicopathological results, which is a preliminary mechanism for the treatment of molecular swelling with sphingosine kinase 1 as a target. The purpose of this study is to explore the purpose of SPH. The role of K1 in the development of bladder urothelial carcinoma and its mechanism provide a theoretical basis for the diagnosis and treatment of SphK1 as a molecular target for the diagnosis and treatment of bladder urothelial carcinoma. Materials, methods and results are based on the phorbol12-myristate13-acetate (phorbol12-myristate13-acetate, PMA) of the 12- myrisate (phorbol12-myristate13-acetate, PMA) of the cells and molecules. Two methyl sphingosine (n-dimethyld-erythrosphingsine, DMS) was a SphK1 inhibitor; 0.9%nacl as a blank reagent, the BIU-87 cell line of bladder cancer was treated as the activation group. The proliferation of the cells in each group was measured by the.Cck-8 method in the inhibition group and the blank control group. The relative invasion rate and mobility of BIU-87 cells in each group were measured by the Transwell compartment model. The levels of Sphk1, ERK1/2 and p-ERK1/2 protein were measured by Western-blot, and the protein content of MMP-2, MMP-9, VEGF and uPA in the supernatant of each group was detected by ELISA. Results: SphK1 activator can promote the invasion and migration of BIU-87 cells, and obviously enhance the protein expression of Sph K1, ERK1/2 and p-ERK1/2, and promote the protein secretion of MMP-2, MMP-9 and u PA, and promote the formation of VM in the three-dimensional culture system constructed Matrigel. Cell invasion and migration, inhibit the protein expression of Sph K1, ERK1/2 and p-ERK1/2, and inhibit the protein secretion of MMP-2, MMP-9 and u PA, and can not form tubular VM in three-dimensional culture, and obviously weaken the expression of VEGF protein. The expression of mRNA and protein and its relationship with the clinicopathological features of bladder cancer were analyzed. Results: the expression of SphK1m RNA in the 37 bladder cancer tissues and adjacent normal tissues was found to be significantly higher than that in the normal tissues adjacent to the carcinoma, and the Sph K1 protein table in the pathological sections of the bladder cancer was found. The 5 year survival rate of the patients with high expression of Sph K1 was significantly decreased (P0.001) by plotting the Kaplan-Meier survival curve (P0.001). The multivariable Cox regression analysis showed that the high expression of SphK1 could be used as an independent adverse prognostic factor of this disease. Conclusion Sph K1 was found in the 5. Bladder urothelial carcinoma plays an important role in the development and development of the bladder urothelial carcinoma, which may be associated with the activation of the signal ERK1/2 pathway to promote the secretion of MMP-2, MMP-9 and u PA protein, thereby promoting the expression of VEGF and enhancing the invasion and migration of tumor cells. We can treat Sph K1 as a diagnosis and treatment of bladder urothelial carcinoma. Molecular targets.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R737.14

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