發(fā)布時間：2018-08-27 17:22

【摘要】：結(jié)直腸癌是嚴(yán)重威脅人類健康的重大疾病。轉(zhuǎn)移是導(dǎo)致結(jié)直腸癌預(yù)后不良的關(guān)鍵問題,超過三分之一的結(jié)直腸癌患者最終會發(fā)生轉(zhuǎn)移,給臨床治療帶來極大困難,嚴(yán)重影響患者的療效和生存質(zhì)量。因此,結(jié)直腸癌治療的難題在于轉(zhuǎn)移,必須尋找可以有效干預(yù)結(jié)直腸癌轉(zhuǎn)移的分子靶點,而這有賴于結(jié)直腸癌轉(zhuǎn)移發(fā)生機制研究的突破。然而,結(jié)直腸癌的轉(zhuǎn)移是涉及多個步驟的連續(xù)過程,極其復(fù)雜,其具體機制目前仍不清楚。腫瘤細(xì)胞之間存在明顯的異質(zhì)性,并非所有腫瘤細(xì)胞都能發(fā)生轉(zhuǎn)移,只有那些獲得一定遷移和侵襲能力的腫瘤細(xì)胞才能突破重重障礙,形成遠(yuǎn)隔組織或器官轉(zhuǎn)移灶。研究表明,上皮-間質(zhì)轉(zhuǎn)化(Epithelial-mesenchymal transition,EMT)是導(dǎo)致腫瘤細(xì)胞發(fā)生侵襲和轉(zhuǎn)移的重要原因,是腫瘤發(fā)生轉(zhuǎn)移的關(guān)鍵啟動步驟。在EMT啟動腫瘤細(xì)胞侵襲轉(zhuǎn)移的過程中,播撒的腫瘤細(xì)胞獲得了類似腫瘤干細(xì)胞自我更新的特性,進(jìn)而形成了肉眼可見的轉(zhuǎn)移灶。近年隨著對腫瘤免疫研究的不斷深入,一組介導(dǎo)免疫調(diào)節(jié)的重要分子——負(fù)性B7家族分子:PD-L1(B7-H1)/PD-1、B7-H3、B7-H4和CTLA-4以及Tim-3等,異常表達(dá)在一系列腫瘤組織和/或免疫細(xì)胞,參與腫瘤免疫逃逸、腫瘤侵襲轉(zhuǎn)移等過程,并與腫瘤臨床病理和預(yù)后密切相關(guān)。其中,抗CTLA-4、PD-1及PD-L1單抗在多種腫瘤治療中顯示出卓著療效,負(fù)性B7家族分子在腫瘤免疫治療中的前景受到空前關(guān)注。B7-H3是2001年從人DC的cDNA庫中克隆得到的一個B7家族成員。人B7-H3基因定位于15q24,屬免疫球蛋白家族的I型跨膜蛋白。迄今為止,已先后發(fā)現(xiàn)B7-H3蛋白在諸多腫瘤組織中高表達(dá),包括胃癌、肺癌、前列腺癌等。B7-H3在人類腫瘤組織的異常表達(dá),并參與了腫瘤的發(fā)生和發(fā)展,但是B7-H3參與腫瘤生長與轉(zhuǎn)移的作用機制尚不完全明確。本研究所一直致力于負(fù)性B7家族分子(PD-L1、B7-H3、B7-H4、CTLA-4和Tim-3)與腫瘤免疫的研究,近來在結(jié)合蛋白研究方面取得部分進(jìn)展,通過質(zhì)譜分析找到了可以與b7-h3相互作用的原癌基因編碼蛋白c-met,鑒此為本課題展開奠定了良好的基礎(chǔ)。c-met在多種腫瘤中高表達(dá)并異常激活,在腫瘤發(fā)生發(fā)展、侵襲轉(zhuǎn)移等多個環(huán)節(jié)均發(fā)揮關(guān)鍵作用。鑒此,本文以人結(jié)直腸癌為研究對象,b7-h3和c-met相互作用為主線,通過臨床標(biāo)本和體內(nèi)外實驗多個層面的研究,進(jìn)一步探討b7-h3調(diào)控結(jié)直腸癌emt促進(jìn)轉(zhuǎn)移的作用機制及臨床意義。第一部分負(fù)性共刺激分子b7-h3在結(jié)直腸癌中的表達(dá)及臨床意義【目的】分析人結(jié)直腸癌細(xì)胞株、人結(jié)直腸癌新鮮組織以及石蠟組織中共刺激分子b7-h3的表達(dá),進(jìn)而探討b7-h3與結(jié)直腸癌臨床病理因素、患者預(yù)后的關(guān)系,揭示b7-h3在結(jié)直腸癌組織標(biāo)本的異常表達(dá)及其臨床意義�！痉椒ā糠謩e從mrna和蛋白水平分析了6株結(jié)直腸癌細(xì)胞株中b7-h3的表達(dá),并同時觀察了腫瘤細(xì)胞上清中可溶性b7-h3的表達(dá);進(jìn)一步比較分析了10例手術(shù)切除的結(jié)直腸癌腫瘤組織和遠(yuǎn)端正常腸組織標(biāo)本中b7-h3的mrna和蛋白水平的表達(dá)差異;進(jìn)而通過免疫組化方法分析了197例配對的結(jié)直腸癌癌組織和癌旁組織中b7-h3的表達(dá),并結(jié)合病理資料及生存時間,統(tǒng)計分析b7-h3表達(dá)的臨床意義�！窘Y(jié)果】(1)6株結(jié)直腸癌細(xì)胞表面均存在不同程度膜型b7-h3的表達(dá),同時還可分泌可溶性b7-h3;(2)較之于遠(yuǎn)端正常腸組織,手術(shù)切除的新鮮結(jié)直腸癌腫瘤組織中b7-h3呈高表達(dá),免疫組化分析石蠟組織結(jié)果顯示:197例結(jié)直腸癌患者中,73例患者組織低表達(dá)b7-h3分子,占37.1%;124例患者組織高表達(dá)b7-h3分子,占62.9%;(3)結(jié)合患者的術(shù)后總體生存時間和無病生存時間進(jìn)行分析,發(fā)現(xiàn)b7-h3表達(dá)的高低與患者的預(yù)后密切相關(guān)(p0.01),較之于b7-h3低表達(dá)的患者,b7-h3高表達(dá)患者的總體生存時間與無病生存時間都大幅下降;(4)進(jìn)一步的統(tǒng)計發(fā)現(xiàn),b7-h3表達(dá)高低與患者的duke's分期相關(guān)(p0.05),而與性別、年齡、腫瘤大小、腫瘤分期和淋巴結(jié)轉(zhuǎn)移等無統(tǒng)計學(xué)差異;(5)隨著b7-h3表達(dá)水平的升高,患者發(fā)生遠(yuǎn)處轉(zhuǎn)移與b7-h3表達(dá)水平的呈相關(guān)性上升。【結(jié)論】共刺激分子b7-h3在結(jié)直腸癌細(xì)胞、新鮮組織以及石蠟組織中異常高表達(dá),并與患者的duke's分期和不良預(yù)后密切相關(guān),提示了b7-h3在結(jié)直腸癌的進(jìn)展與轉(zhuǎn)移中發(fā)揮重要作用。第二部分b7-h3在結(jié)直腸癌細(xì)胞的emt及干性維持中的作用【目的】通過分析b7-h3對腫瘤細(xì)胞遷移、侵襲能力以及干性的作用,以期明確b7-h3在結(jié)直腸癌細(xì)胞emt和干性維持中的調(diào)控作用�！痉椒ā恳愿弑磉_(dá)b7-h3的結(jié)直腸癌細(xì)胞系hct116、sw480作為研究對象,經(jīng)sirna干擾下調(diào)b7-h3的表達(dá)后,通過real-timepcr、westernblot和免疫熒光法觀察腫瘤細(xì)胞中emt相關(guān)標(biāo)志分子、轉(zhuǎn)錄因子和干性基因的mrna和蛋白水平表達(dá)的變化;進(jìn)一步以沉默或過表達(dá)b7-h3的hct116、sw480為研究對象,通過遷移實驗、劃痕實驗和侵襲實驗觀察腫瘤細(xì)胞體外遷移和侵襲的能力;通過體外細(xì)胞球?qū)嶒灪突�療藥抵抗實�?以及皮下接種腫瘤模型分析b7-h3在腫瘤細(xì)胞干性維持中的作用�！窘Y(jié)果】(1)b7-h3表達(dá)下調(diào)后,結(jié)直腸癌細(xì)胞hct116、sw480中的上皮標(biāo)志e-cadherin呈上調(diào)表達(dá),間質(zhì)標(biāo)志vimentin和n-cadherin呈下調(diào)表達(dá),且emt相關(guān)轉(zhuǎn)錄因子snail、slug、zeb1、twist的表達(dá)也顯著下調(diào);(2)b7-h3表達(dá)下調(diào)后,腫瘤細(xì)胞中干性基因(cd133、nanog、bmi1、lgr5、abcb1、abcg2、oct4)呈下調(diào)表達(dá);(3)進(jìn)一步的遷移實驗、劃痕實驗和侵襲實驗結(jié)果顯示,b7-h3表達(dá)下調(diào)后,結(jié)直腸癌細(xì)胞hct116、sw480的體外遷移和侵襲能力受到抑制;但過表達(dá)b7-h3后,結(jié)直腸癌細(xì)胞hct116、sw480的體外遷移和侵襲能力又得到增強;(4)體外細(xì)胞球?qū)嶒灠l(fā)現(xiàn):b7-h3表達(dá)下調(diào)后,結(jié)直腸癌細(xì)胞hct116、sw480的細(xì)胞球形成能力受到抑制;(5)化療藥抵抗實驗進(jìn)一步揭示了b7-h3表達(dá)下調(diào)后,結(jié)直腸癌細(xì)胞hct116、sw480的對化療藥更為敏感;(6)通過荷瘤小鼠體內(nèi)實驗證實,結(jié)直腸癌細(xì)胞hct116中b7-h3表達(dá)下調(diào)后,其在小鼠體內(nèi)形成腫瘤的體積及數(shù)量均顯著降低�！窘Y(jié)論】共刺激分子b7-h3表達(dá)沉默后,結(jié)直腸癌細(xì)胞的間質(zhì)化轉(zhuǎn)變受阻,進(jìn)而腫瘤細(xì)胞的體外遷移和侵襲能力受到抑制。同時,結(jié)直腸癌細(xì)胞的干性標(biāo)志表達(dá)也顯著下調(diào),進(jìn)而其體外成球和化療藥抵抗能力減弱。此外,結(jié)直腸癌細(xì)胞的體內(nèi)成瘤能力也顯著降低。第三部分b7-h3、e-cadherin及c-met在結(jié)直腸癌中的共表達(dá)及臨床意義【目的】在發(fā)現(xiàn)了b7-h3可與原癌基因編碼蛋白c-met相互作用,以及明確了b7-h3對emt調(diào)控作用的基礎(chǔ)上,通過分析結(jié)直腸癌組織中b7-h3、上皮標(biāo)志e-cadherin及c-met的表達(dá),探討三者與結(jié)直腸癌臨床病理因素、患者預(yù)后的關(guān)系,揭示b7-h3、e-cadherin及c-met共表達(dá)在結(jié)直腸癌發(fā)生發(fā)展及預(yù)后中的作用�！痉椒ā渴占�197例手術(shù)切除的結(jié)直腸癌癌組織和癌旁組織,通過免疫組化方法分析了197例配對組織中b7-h3、e-cadherin及c-met的表達(dá),進(jìn)一步結(jié)合病理資料及生存時間,統(tǒng)計分析b7-h3、e-cadherin及c-met表達(dá)的相關(guān)性及臨床意義�！窘Y(jié)果】(1)免疫組化結(jié)果顯示,197例結(jié)直腸癌患者中,31.5%組織標(biāo)本中e-cadherin正常表達(dá)于細(xì)胞膜,而68.5%組織標(biāo)本中e-cadherin異常表達(dá)于細(xì)胞漿或不表達(dá);(2)71.1%組織標(biāo)本中異常表達(dá)c-met,28.9%組織標(biāo)本中不表達(dá)c-met;(3)進(jìn)一步統(tǒng)計發(fā)現(xiàn),結(jié)直腸癌腫瘤組織中異常表達(dá)的b7-h3和膜e-cadherin之間呈負(fù)相關(guān)的關(guān)系,且高表達(dá)b7-h3和漿e-cadherin的患者的總體生存時間與無病生存時間都大幅下降;(4)異常表達(dá)的b7-h3和c-met之間呈正相關(guān)的關(guān)系,且具有顯著統(tǒng)計學(xué)差異�！窘Y(jié)論】在結(jié)直腸癌腫瘤組織中異常高表達(dá)b7-h3、漿e-cadherin和c-met,b7-h3與膜e-cadherin之間呈負(fù)相關(guān),與c-met之間呈正相關(guān),顯著影響患者的總體生存時間和無病生存時間。這些均提示b7-h3與c-met相互作用后可參與結(jié)直腸癌emt的調(diào)控。第四部分b7-h3通過c-met途徑對結(jié)直腸癌細(xì)胞emt的調(diào)控機制【目的】通過探討b7-h3與c-met相互作用后在結(jié)直腸癌細(xì)胞emt中的作用機制,為b7-h3促進(jìn)人結(jié)直腸癌的進(jìn)展和轉(zhuǎn)移提供理論依據(jù)�！痉椒ā恳赃^表達(dá)b7-h3的結(jié)直腸癌細(xì)胞系sw480作為研究對象,通過免疫共沉淀和westernblot方法觀察結(jié)直腸癌細(xì)胞中b7-h3和c-met的相互作用;通過共聚焦顯微鏡觀察結(jié)直腸癌細(xì)胞中b7-h3和c-met的共定位。以低表達(dá)b7-h3的結(jié)直腸癌細(xì)胞rko為研究對象,進(jìn)行b7-h3過表達(dá)和c-met沉默共處理后,通過遷移實驗和劃痕實驗分析b7-h3/c-met對腫瘤細(xì)胞遷移侵襲能力的調(diào)控作用,通過干性基因檢測和細(xì)胞球?qū)嶒炋接慴7-h3/c-met對腫瘤細(xì)胞干性維持的影響。在此基礎(chǔ)上,外源加入人重組b7-h3蛋白觀察c-met、p-met的表達(dá)變化;進(jìn)一步通過westernblot觀察b7-h3表達(dá)沉默后結(jié)直腸癌細(xì)胞中c-met下游關(guān)鍵信號分子akt、erk磷酸化水平以及emt轉(zhuǎn)錄因子snail的表達(dá)變化�！窘Y(jié)果】(1)在過表達(dá)b7-h3的sw480細(xì)胞中共刺激分子b7-h3可與c-met相關(guān)作用,通過共聚焦顯微鏡可觀察到兩者存在共定位;(2)較之于對照組,b7-h3過表達(dá)組的腫瘤細(xì)胞表面干性標(biāo)志表達(dá)明顯上調(diào),且遷移、劃痕愈合和細(xì)胞球形成能力顯著增強;而較之于b7-h3過表達(dá)組,b7-h3過表達(dá)/c-met沉默表達(dá)共處理組的腫瘤細(xì)胞表面干性標(biāo)志表達(dá)明顯下調(diào),同時遷移、劃痕愈合和細(xì)胞球形成能力也顯著減弱;(3)外源加入的人重組蛋白b7-h3可上調(diào)p-met的表達(dá)水平;(4)當(dāng)b7-h3表達(dá)沉默后會影響下游關(guān)鍵信號分子akt、erk的磷酸化水平以及emt關(guān)鍵轉(zhuǎn)錄因子snail的表達(dá)�！窘Y(jié)論】共刺激分子b7-h3可與c-met相互作用,其后通過影響p-met的表達(dá),進(jìn)而通過下游信號軸akt/snail參與調(diào)控結(jié)直腸癌細(xì)胞的emt,從而影響腫瘤的轉(zhuǎn)移。綜上所述,本課題通過臨床標(biāo)本分析和體內(nèi)外多個層面和不同水平的實驗證實共刺激分子b7-h3與結(jié)直腸癌臨床病理、進(jìn)展和轉(zhuǎn)移密切相關(guān),繼而發(fā)現(xiàn)b7-h3通過emt機制加速結(jié)直腸癌細(xì)胞發(fā)生間質(zhì)化轉(zhuǎn)變,進(jìn)而促進(jìn)腫瘤的進(jìn)展與轉(zhuǎn)移,b7-h3分子有望成為監(jiān)測結(jié)直腸癌轉(zhuǎn)移的有價值的生物標(biāo)志。更為重要的是本文首次發(fā)現(xiàn)結(jié)直腸癌細(xì)胞表達(dá)b7-h3可以與原癌基因編碼蛋白c-met相互作用,促進(jìn)c-Met信號及相應(yīng)生物學(xué)功能,進(jìn)而闡明了B7-H3調(diào)控結(jié)直腸癌EMT進(jìn)程促進(jìn)腫瘤進(jìn)展與轉(zhuǎn)移的作用機制,為結(jié)直腸癌轉(zhuǎn)移的免疫干預(yù)提供一個新的靶點。
[Abstract]:Colorectal cancer is a serious threat to human health. Metastasis is a key problem leading to poor prognosis of colorectal cancer. More than one third of colorectal cancer patients will eventually metastasize, which brings great difficulties to clinical treatment and seriously affects the efficacy and quality of life of patients. However, the metastasis of colorectal cancer is a continuous process involving multiple steps, and its specific mechanism is still unclear. There is obvious heterogeneity between tumor cells, not all tumor details. Only those tumor cells with certain ability of migration and invasion can break through barriers and form distant tissue or organ metastasis foci. Studies have shown that epithelial-mesenchymal transition (EMT) is an important cause of invasion and metastasis of tumor cells, and metastasis of tumor. In the process of EMT initiating invasion and metastasis of tumor cells, seeded tumor cells acquire self-renewal characteristics similar to that of tumor stem cells, thus forming metastatic foci visible to the naked eye. L1 (B7-H1) / PD-1, B7-H3, B7-H4, CTLA-4 and Tim-3 are abnormally expressed in a series of tumor tissues and / or immune cells, which are involved in tumor immune escape, tumor invasion and metastasis, and are closely related to the clinical pathology and prognosis of the tumor. Among them, anti-CTLA-4, PD-1 and PD-L1 monoclonal antibodies have shown remarkable therapeutic effects in a variety of tumor treatments, and negative B7 have been found. B7-H3 is a member of the B7 family cloned from the human DC cDNA Library in 2001. The human B7-H3 gene is located at 15q24 and belongs to the immunoglobulin family I transmembrane protein. B7-H3 is abnormally expressed in human tumor tissues and participates in the occurrence and development of tumor. However, the mechanism of B7-H3 involved in tumor growth and metastasis is still unclear. Some progress has been made in this field. The proto-oncogene coding protein c-met, which can interact with b7-h3, has been found by mass spectrometry analysis. It has laid a good foundation for this research. c-met is highly expressed and abnormally activated in various tumors, and plays a key role in the development, invasion and metastasis of tumors. The interaction between B7-H3 and c-Met in human colorectal cancer was the main clue. the mechanism and clinical significance of B7-H3 regulating the metastasis of colorectal cancer by EMT were further explored through clinical specimens and in vivo and in vitro experiments. part one: the expression and clinical significance of negative costimulatory molecule B7-H3 in colorectal cancer [Methods] To analyze the expression of costimulatory molecule B7-H3 in human colorectal cancer cell lines, fresh colorectal cancer tissues and paraffin tissues, and to explore the relationship between B7-H3 and clinicopathological factors, prognosis of colorectal cancer patients, and to reveal the abnormal expression of B7-H3 in colorectal cancer tissues and its clinical significance. The expression of B7-H3 was analyzed in 6 colorectal cancer cell lines, and the expression of soluble B7-H3 in the supernatant of tumor cells was also observed. The expression of B7-H3 mRNA and protein in 10 colorectal cancer tissues and distal normal intestinal tissues were compared and analyzed by immunohistochemistry. The expression of B7-H3 in colorectal cancer tissues and adjacent tissues of 97 matched patients was analyzed by pathological data and survival time. The expression of B7-H3 was high in fresh colorectal cancer tissues after operation. The results of immunohistochemical analysis showed that in 197 colorectal cancer patients, 73 cases had low expression of b7-h3, accounting for 37.1%; 124 cases had high expression of b7-h3, accounting for 62.9%; (3) combined with the overall survival time and disease-free survival time of the patients after operation. It was found that the expression of B7-H3 was closely related to the prognosis of patients (p0.01). Compared with the patients with low expression of b7-h3, the overall survival time and disease-free survival time of patients with high expression of B7-H3 decreased significantly. (4) Further statistics showed that the expression of B7-H3 was related to the duke's stage of patients (p0.05), but with gender, age, tumor size, and so on. There was no significant difference in tumor stage and lymph node metastasis. (5) With the increase of B7-H3 expression level, distant metastasis and B7-H3 expression level were significantly correlated. Part two: the role of B7-H3 in EMT and dry maintenance of colorectal cancer cells [Methods] The expression of EMT-related markers, transcription factors and dry gene mRNA and protein in colorectal cancer cell lines HCT116 and SW480 were detected by real-time pcr, Western blot and immunofluorescence after siRNA interference. One step was to observe the ability of tumor cells to migrate and invade in vitro by migration test, scratch test and invasion test, and to analyze the role of B7-H3 in the maintenance of tumor cell stem by cell ball test in vitro, chemotherapeutic drug resistance test and subcutaneous inoculation tumor model. Results: (1) after B7-H3 expression was down-regulated, the expression of e-cadherin, the epithelial marker of HCT116 and sw480, vimentin and n-cadherin, and the expression of snail, slug, ZEB1 and twist were down-regulated significantly; (2) after B7-H3 expression was down-regulated, the trunk genes (cd133, nanog, bmi1, lgr5, ab) were down-regulated. Cb1, abcg2, Oct4 were down-regulated; (3) further migration test, scratch test and invasion test showed that the down-regulated expression of B7-H3 inhibited the migration and invasion ability of colorectal cancer cells hct116, SW480 in vitro; but after overexpression of b7-h3, the migration and invasion ability of colorectal cancer cells hct116, SW480 in vitro was enhanced; (4) in vitro migration and invasion ability of colorectal cancer cells hct116, SW480 was enhanced; (4) in vitro migration and invasion ability of colorectal cancer cells SW48 The results of cell sphere assay showed that the sphere formation ability of colorectal cancer cells HCT116 and SW480 was inhibited after the down-regulation of B7-H3 expression; (5) chemotherapeutic drug resistance assay further revealed that after the down-regulation of B7-H3 expression, colorectal cancer cells HCT116 and SW480 were more sensitive to chemotherapeutic drugs; (6) in vivo experiments in tumor-bearing mice confirmed that B in colorectal cancer cells HCT116 was more sensitive to chemotherapeutic drugs. [Conclusion] When the expression of costimulatory molecule B7-H3 was silenced, the stromal transformation of colorectal cancer cells was blocked, and the migration and invasion of colorectal cancer cells were inhibited in vitro. The co-expression of b7-h3, E-cadherin and c-Met in colorectal cancer and its clinical significance [Objective] It was found that B7-H3 could interact with c-met, a proto-oncogene coding protein, and the relationship between B7-H3 and EMT was clarified. On the basis of regulation, the expression of b7-h3, E-cadherin and c-Met in colorectal cancer tissues was analyzed to explore the relationship between the expression of b7-h3, E-cadherin and c-Met and the clinicopathological factors and prognosis of colorectal cancer. The role of co-expression of b7-h3, E-cadherin and c-Met in the occurrence, development and prognosis of colorectal cancer was revealed. The expressions of b7-h3, E-cadherin and c-Met in 197 matched tissues of colorectal cancer and adjacent tissues were analyzed by immunohistochemistry. The correlation and clinical significance of the expressions of b7-h3, E-cadherin and c-Met were analyzed by combining pathological data and survival time. 31.5% of the tissue specimens showed normal expression of E-cadherin in the cell membrane, while 68.5% of the tissue specimens showed abnormal expression of E-cadherin in the cytoplasm or not; (2) 71.1% of the tissue specimens showed abnormal expression of c-met, 28.9% of the tissue specimens did not express c-met; (3) further statistics showed that abnormal expression of b7-h 3 and E-cadherin in the colorectal cancer tissue were found. A negative correlation, and the overall survival time of B7-H3 and high expression of E-cadherin in plasma of patients with disease-free survival time decreased significantly; (4) there is a positive correlation between the abnormal expression of B7-H3 and c-Met, and the difference was statistically significant. [Conclusion] in colorectal tumor tissue expression B7-H3, E-cadherin and c-Met pulp And there was a negative correlation between B7-H3 and membrane E-cadherin was positively correlated with c-Met, significantly affect the overall survival time and disease-free survival time. All these results indicate that B7-H3 interacts with c-met can be involved in the regulation of colorectal cancer EMT. The fourth part of B7-H3 via c-met pathway on colorectal cancer cell regulatory mechanisms of EMT [Objective] through To explore the mechanism in colorectal cancer cells in EMT B7-H3 and c-Met interaction, and provide a theoretical basis for the B7-H3 to promote the progression and metastasis of human colorectal cancer. [Methods] the expression of colorectal cancer cell line SW480 B7-H3 as the research object, by CO immunoprecipitation and Westernblot method to observe the B7-H3 and C-M rectal cancer cells The interaction of ET; B7-H3 and c-Met observe the co localization of node of colorectal carcinoma cells by confocal microscopy. The low expression of RKO on colorectal cancer cell B7-H3 as the research object, B7-H3 overexpression and silencing of c-met after incubated by migration assay and scratch test analysis of regulation effect of b7-h3/c-met on invasion and migration of tumor cells, Through dry gene detection and cell ball experiment to explore the effects of b7-h3/c-met on tumor cell stemness. On this basis, exogenous recombinant human B7-H3 protein was observed in c-Met, the expression of p-met; further observed by Westernblot B7-H3 after silencing the expression of c-Met in colorectal cancer cells downstream of key signaling molecule Akt phosphorylation of ERK in water Changes and expression of EMT transcription factor Snail. [results] (1) the over expression of B7-H3 in the SW480 cell stimulatory molecule B7-H3 can be associated with c-Met, by confocal microscopy can be observed between the two co localization; (2) compared with control group, B7-H3 overexpression group of tumor cell surface marker expression dry increased obviously, and the migration, Wound healing and cell sphere formation ability is significantly improved; and compared with B7-H3 overexpression group, B7-H3 overexpression of /c-met silencing tumor cell surface marker dry treated group was significantly reduced, while migration, wound healing and cell sphere formation ability also significantly reduced; (3) human recombinant protein B7-H3 exogenous could increase p-met table Reached the level; (4) when B7-H3 knockdown affects key signaling molecules downstream of Akt, ERK phosphorylation and EMT expression of key transcription factor Snail. [Conclusion] the costimulatory molecule B7-H3 interacts with c-Met, followed by the expression of p-met, and then through the downstream signal axis akt/snail is involved in the regulation of colorectal cancer cells emt, Which affect the metastasis of tumor. In summary, this topic through the analysis of clinical specimens and in vivo and in many aspects and different levels of experiment
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R735.34

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