本文選題：胃癌 + 耐藥　； 參考：《第四軍醫(yī)大學(xué)》2017年博士論文

【摘要】：【背景】胃癌是最常見的消化道惡性腫瘤,化療在其綜合治療中發(fā)揮著極其重要的作用。但是,在臨床上存在著化療藥物可以使腫瘤迅速縮小,但對(duì)患者生存期延長不明顯的悖論。造成這一現(xiàn)象的重要原因是腫瘤細(xì)胞的多藥耐藥及其導(dǎo)致的腫瘤細(xì)胞惡性表型的進(jìn)展。既往有研究發(fā)現(xiàn)化療藥物可以通過誘導(dǎo)腫瘤細(xì)胞發(fā)生EMT、改變腫瘤微環(huán)境以及富集腫瘤干細(xì)胞等途徑促進(jìn)多種腫瘤的侵襲轉(zhuǎn)移。但是,化療在促進(jìn)胃癌細(xì)胞侵襲轉(zhuǎn)移中的作用研究尚不充分。分泌蛋白質(zhì)組學(xué)是以分泌和脫落的細(xì)胞膜蛋白為主要研究對(duì)象的蛋白質(zhì)組學(xué)重要分支,其在腫瘤研究中的應(yīng)用極大的推動(dòng)新型腫瘤標(biāo)志物的發(fā)現(xiàn),也為闡明腫瘤發(fā)生發(fā)展的機(jī)制提供了新的技術(shù)手段�！灸康摹客ㄟ^分泌蛋白質(zhì)組學(xué)方法篩選促進(jìn)胃癌多藥耐藥細(xì)胞侵襲轉(zhuǎn)移的候選分子并明確其作用機(jī)制【方法】1.采用Transwell和裸鼠尾靜脈注射肺部轉(zhuǎn)移實(shí)驗(yàn)檢測(cè)胃癌多藥耐藥和親本細(xì)胞侵襲轉(zhuǎn)移能力的差別;采用非標(biāo)定量蛋白質(zhì)譜技術(shù)篩選胃癌耐藥和親本細(xì)胞無血清條件培養(yǎng)基中差異分泌蛋白,并與既往基因芯片篩選結(jié)果對(duì)比分析取交集;采用q RT-PCR和Western blot技術(shù)對(duì)篩選結(jié)果在胃癌及其他腫瘤的耐藥細(xì)胞系中進(jìn)行驗(yàn)證;采用公共數(shù)據(jù)庫查詢候選分子在腫瘤中表達(dá)的臨床意義;在胃癌細(xì)胞系和PDX裸鼠模型中檢測(cè)化療藥物能否誘導(dǎo)候選分子表達(dá)。2.采用Transwell和尾靜脈注射體內(nèi)轉(zhuǎn)移實(shí)驗(yàn)在耐藥和親本胃癌細(xì)胞中檢測(cè)下調(diào)和過表達(dá)ADAM22對(duì)細(xì)胞侵襲轉(zhuǎn)移能力的影響。3.采用q RT-PCR和Western blot技術(shù)檢測(cè)EMT經(jīng)典標(biāo)志物的表達(dá);采用體外黏附實(shí)驗(yàn)檢測(cè)干預(yù)ADAM22表達(dá)對(duì)細(xì)胞對(duì)細(xì)胞外基質(zhì)Fibronectin和CollagenⅠ黏附能力的變化并采用免疫熒光技術(shù)檢測(cè)黏著斑標(biāo)志物和F-actin纖維形成情況;采用尾靜脈注射熒光標(biāo)記腫瘤細(xì)胞技術(shù)檢測(cè)干預(yù)ADAM22對(duì)腫瘤細(xì)胞在裸鼠肺內(nèi)駐留能力的影響;采用體外穿內(nèi)皮實(shí)驗(yàn)檢測(cè)干預(yù)ADAM22表達(dá)對(duì)腫瘤細(xì)胞穿過內(nèi)皮細(xì)胞能力的影響;采用Matrigel-on-Top(Mo T)3D培養(yǎng)技術(shù)檢測(cè)干預(yù)ADAM22對(duì)單個(gè)腫瘤細(xì)胞克隆形成及絲狀偽足樣凸起(FLPs)形成能力的影響。4.采用Western blot技術(shù)檢測(cè)下調(diào)ADAM22表達(dá)對(duì)黏附相關(guān)分子和Integrinβ1(ITGB1)表達(dá)和活性的影響;采用GST-pulldown聯(lián)合Western blot技術(shù)檢測(cè)下調(diào)ADAM22表達(dá)對(duì)胃癌耐藥細(xì)胞中Rac1和Cdc42活性的影響;在下調(diào)ADAM22表達(dá)的耐藥細(xì)胞中分別檢測(cè)ITGB1激活抗體對(duì)細(xì)胞黏附、體內(nèi)外侵襲遷移能力的影響;采用免疫共沉淀聯(lián)合Western blot技術(shù)檢測(cè)野生及突變型ADAM22與ITGB1的相互作用;采用針對(duì)ADAM22 m RNA 5’-UTR的si RNA下調(diào)內(nèi)源性ADAM22表達(dá)后,驗(yàn)證野生和突變型ADAM22對(duì)耐藥細(xì)胞黏附、遷移和3D克隆形成能力的補(bǔ)救作用。5.采用MTT法檢測(cè)下調(diào)ADAM22、Rac1及Cdc42后耐藥細(xì)胞針對(duì)多種化療藥物IC50的變化;采用q RT-PCR和Western blot技術(shù)檢測(cè)檢測(cè)下調(diào)ADAM22、Rac1和Cdc42后HIF1α和ABCB1 m RNA及其編碼蛋白表達(dá)水平變化。6.采用免疫組織化學(xué)染色技術(shù)檢測(cè)ADAM22和HUTS4在胃癌組織原發(fā)灶和轉(zhuǎn)移灶中的表達(dá)情況;采用非參數(shù)統(tǒng)計(jì)方法分析ADAM22在胃癌原發(fā)灶和轉(zhuǎn)移灶中表達(dá)強(qiáng)度的變化;采用Spearman秩相關(guān)分析ADAM22和HUTS4在胃癌組織中表達(dá)的相關(guān)性;采用Kaplan-Meier生存分析和Cox回歸模型分析ADAM22和HUTS4在胃癌組織中表達(dá)的臨床意義。【結(jié)果】1.Transwell和尾靜脈注射實(shí)驗(yàn)結(jié)果表明胃癌耐藥細(xì)胞SGC7901/ADR和SGC7901/VCR的體內(nèi)外侵襲轉(zhuǎn)移能力明顯增強(qiáng);同時(shí),在尾靜脈轉(zhuǎn)移實(shí)驗(yàn)中我們發(fā)現(xiàn),耐藥細(xì)胞不僅成瘤率高,而且轉(zhuǎn)移灶的數(shù)目和體積也較親本細(xì)胞明顯升高。非標(biāo)定量質(zhì)譜鑒定了91個(gè)在2株耐藥細(xì)胞無血清條件培養(yǎng)基中含量明顯升高的分泌蛋白,通過與既往基因芯片結(jié)果對(duì)比發(fā)現(xiàn)其中23個(gè)m RNA水平也明顯升高。q RT-PCR和Western blot驗(yàn)證結(jié)果證實(shí)了ADAM22在2株胃癌耐藥細(xì)胞以及K562和口腔鱗癌等多種腫瘤的耐藥細(xì)胞中表達(dá)升高。通過查詢Kaplan-Meier Plot數(shù)據(jù)庫我們發(fā)現(xiàn)ADAM22高表達(dá)與胃癌患者預(yù)后不良相關(guān)。q RT-PCR和免疫組化結(jié)果表明多種化療藥物可以誘導(dǎo)胃癌細(xì)胞系和PDX腫瘤組織表達(dá)ADAM22。2.Transwell和尾靜脈注射實(shí)驗(yàn)結(jié)果表明下調(diào)ADAM22表達(dá)后胃癌耐藥細(xì)胞SGC7901/ADR和SGC7901/VCR的體內(nèi)外侵襲轉(zhuǎn)移能力受到明顯抑制。特別需要指出的是在尾靜脈注射實(shí)驗(yàn)中耐藥細(xì)胞不僅成瘤率明顯下降,腫瘤轉(zhuǎn)移灶的數(shù)量和尺寸也明顯下降。3.q RT-PCR和Western blot結(jié)果表明EMT經(jīng)典標(biāo)志物E-cadherin和Vimentin表達(dá)不受ADAM22影響。下調(diào)ADAM22后耐藥細(xì)胞SGC7901/ADR和SGC7901/VCR對(duì)細(xì)胞外基質(zhì)蛋白Fibronectin和CollagenⅠ的黏附能力明顯下降,在肺部駐留能力也受到顯著抑制。免疫熒光結(jié)果表明下調(diào)ADAM22后耐藥細(xì)胞黏著斑和F-actin纖維形成受到明顯抑制。同時(shí),耐藥細(xì)胞的體外內(nèi)皮穿過能力和Mo T培養(yǎng)條件下的克隆形成能力和FLPs形成數(shù)量也明顯下降。4.下調(diào)ADAM22表達(dá)后胃癌耐藥細(xì)胞中黏附相關(guān)分子FAK以及Paxillin的磷酸化水平受到了明顯抑制。GST-pulldown聯(lián)合Western blot結(jié)果表明干擾ADAM22表達(dá)后耐藥細(xì)胞中Rho GTP酶Rac1和Cdc42的活性也明顯下降。針對(duì)ITGB1活化表位HUTS4的Western blot結(jié)果顯示,ADAM22表達(dá)下降后ITGB1的活性也隨之下降。功能實(shí)驗(yàn)表明激活I(lǐng)TGB1可以逆轉(zhuǎn)下調(diào)ADAM22對(duì)耐藥細(xì)胞黏附和體內(nèi)外侵襲遷移能力的抑制。免疫共沉淀結(jié)果表明ITGB1可以與野生型ADAM22相互作用,但不能與Disintegrin缺失的突變型ADAM22相互作用。野生型而非突變型ADAM22可以補(bǔ)救下調(diào)內(nèi)源性ADAM22對(duì)胃癌耐藥細(xì)胞黏附、遷移和3D克隆形成能力的抑制。5.在胃癌耐藥細(xì)胞中下調(diào)ADAM22、Rac1或者Cdc42均可以顯著降低SGC7901/ADR和SGC7901/VCR對(duì)多種化療藥物的IC50。q RT-PCR和Western blot結(jié)果顯示在胃癌耐藥細(xì)胞中下調(diào)ADAM22或者Rac1和Cdc42的表達(dá)可以顯著降低HIF1α和ABCB1及其編碼蛋白的表達(dá)。6.ADAM22在胃癌轉(zhuǎn)移灶中的表達(dá)水平明顯高于其相應(yīng)的原發(fā)灶組織。相關(guān)性分析表明ADAM22在胃癌組織中的表達(dá)與HUTS4的表達(dá)呈正相關(guān)。Kaplan-Meier生存分析顯示胃癌組織中ADAM22和HUTS4高表達(dá)與患者預(yù)后差相關(guān)。Cox多因素回歸分析發(fā)現(xiàn)ADAM22或HUTS4高表達(dá)及腫瘤的N分期和患者年齡是患者預(yù)后不良的獨(dú)立風(fēng)險(xiǎn)預(yù)測(cè)因子。【結(jié)論】本課題篩選并闡明了一條促進(jìn)胃癌耐藥細(xì)胞侵襲轉(zhuǎn)移的信號(hào)通路:化療藥物誘導(dǎo)胃癌細(xì)胞高表達(dá)ADAM22,后者通過Disintegrin結(jié)構(gòu)激活I(lǐng)TGB1增加腫瘤細(xì)胞對(duì)細(xì)胞外基質(zhì)的黏附和內(nèi)皮穿透能力,并賦予轉(zhuǎn)移腫瘤細(xì)胞在轉(zhuǎn)移灶微環(huán)境的克隆增殖優(yōu)勢(shì),從而促進(jìn)耐藥細(xì)胞的侵襲轉(zhuǎn)移。同時(shí),該通路通過影響HIF1α和ABCB1及其編碼蛋白的表達(dá)促進(jìn)胃癌細(xì)胞的多藥耐藥。該研究加深了對(duì)胃癌細(xì)胞耐藥和轉(zhuǎn)移關(guān)系的認(rèn)識(shí),并為開發(fā)同時(shí)針對(duì)胃癌耐藥和轉(zhuǎn)移的治療策略提供了靶點(diǎn)。
[Abstract]:[background] gastric cancer is the most common malignant tumor of the digestive tract. Chemotherapy plays an extremely important role in its comprehensive treatment. However, there is a clinical presence of chemotherapeutic drugs that can make the tumor reduce rapidly, but the paradox of prolonging the life period is not obvious. The important reason for this phenomenon is the multidrug resistance and its guidance of the tumor cells. Progress in the malignant phenotype of tumor cells. Previous studies have found that chemotherapeutic agents can promote the invasion and metastasis of various tumors by inducing EMT, changing tumor microenvironment and enriching tumor stem cells. However, the role of chemotherapy in promoting invasion and metastasis of gastric cancer cells is not sufficient. Learning is an important branch of proteomics, which is the main research object of secretory and exudate cell membrane proteins. Its application in cancer research greatly promotes the discovery of new tumor markers and provides new technical means to elucidate the mechanism of tumor development. The candidate molecules of the invasion and metastasis of cancer multidrug resistant cells and the mechanism of its action [method] 1. Transwell and nude mice tail vein were injected into the lungs to detect the difference in the ability of multidrug resistance and the invasion and metastasis of the parent cells. The non standard quantitative protein mass spectrometry was used to screen the gastric cancer resistance and the serum-free conditional culture of the parent cells. The differentially secreted proteins in the substrate were compared with the previous gene chip screening results. Q RT-PCR and Western blot were used to verify the screening results in the drug-resistant cell lines of gastric cancer and other tumors; the clinical significance of the candidate molecules in the tumor was inquired by the public database, and the gastric cancer cell line and the PDX nude mice were used. Whether or not the chemotherapeutic drugs can induce the expression of the candidate molecules to the expression of.2., the effect of down regulation and overexpression of ADAM22 on the invasion and metastasis of cells in the drug-resistant and parent gastric cancer cells was detected by Transwell and caudal vein injection..3. was used to detect the expression of the classical EMT markers using Q RT-PCR and Western blot technology. The effect of ADAM22 expression on the adhesion ability of cell to extracellular matrix Fibronectin and Collagen I was detected by experimental detection, and immunofluorescence technique was used to detect the formation of plaque marker and F-actin fiber. Tail vein injection of fluorescence labeled tumor cell technique was used to detect the ability of ADAM22 to reside in the lung of nude mice. The effect of ADAM22 expression on the ability of tumor cells passing through endothelial cells in vitro, Matrigel-on-Top (Mo T) 3D culture technique was used to detect the influence of intervention ADAM22 on the formation of single tumor cell clone formation and the influence of filamopopfoot like protruding (FLPs) formation energy,.4. using Western blot technique to detect down regulation ADAM22 The effect of expression on the expression and activity of adhesion related molecules and Integrin beta 1 (ITGB1); the effect of GST-pulldown combined with Western blot on the activity of Rac1 and Cdc42 in gastric cancer resistant cells by down regulation of ADAM22; detection of adhesion to cells by ITGB1 activated antibodies and invasion and migration in vivo and in vivo The interaction between wild and mutant ADAM22 and ITGB1 was detected by immunoprecipitation combined with Western blot, and Si RNA for ADAM22 m RNA 5 '-UTR down regulated endogenous ADAM22 expression was used to verify the remedial action of wild and mutant ADAM22 on the adhesion, migration and clone formation of drug-resistant cells. The changes in ADAM22, Rac1 and Cdc42 were measured against a variety of chemotherapeutic agents, IC50, and Q RT-PCR and Western blot techniques were used to detect the changes in ADAM22, Rac1 and Cdc42, and the changes in the expression level of HIF1 alpha and its encoded protein. The expression of ADAM22 in the primary and metastatic foci of gastric cancer was analyzed by non parametric statistical method. The correlation of expression of ADAM22 and HUTS4 in gastric cancer tissues was analyzed by Spearman rank correlation, and the expression of ADAM22 and HUTS4 in gastric cancer tissues was analyzed by Kaplan-Meier survival analysis and Cox regression model. Clinical significance. [results] the results of 1.Transwell and tail vein injection showed that the invasion and metastasis of gastric cancer cells SGC7901/ADR and SGC7901/VCR increased significantly in vivo and in vitro. At the same time, we found that in the tail vein metastasis experiment, the drug resistant cells not only have high tumor formation rate, but also the number and volume of the transfer focus are also significantly higher than those of the parent cells. Non standard quantitative mass spectrometry was used to identify 91 secretory proteins which were significantly elevated in the serum-free medium of 2 resistant cells. By comparison with previous gene chip results, 23 m RNA levels were also significantly increased by.Q RT-PCR and Western blot verification results confirmed that ADAM22 was in 2 gastric cancer resistant cells and K562 and oral squamous cell carcinoma. The expression of multidrug-resistant cells in a variety of tumors was elevated. By querying the Kaplan-Meier Plot database, we found that the high expression of ADAM22 was associated with poor prognosis in patients with gastric cancer,.Q RT-PCR and immunohistochemical results showed that a variety of chemotherapeutic drugs could induce the expression of ADAM22.2.Transwell in gastric cancer cell lines and PDX tumor tissue and the result of the tail vein injection. The invasion and metastasis of gastric cancer resistant cells SGC7901/ADR and SGC7901/VCR were obviously inhibited after the ADAM22 expression was down-regulation. It was particularly pointed out that the tumor rate decreased significantly in the caudal vein injection experiment, and the number and size of tumor metastasis also decreased significantly.3.q RT-PCR and Western blot results indicated EMT classics. The expression of E-cadherin and Vimentin was not affected by ADAM22. After the downregulation of ADAM22, the adhesion ability of SGC7901/ADR and SGC7901/VCR to the extracellular matrix protein Fibronectin and Collagen I was significantly decreased, and the ability to reside in the lungs was also significantly inhibited. The immunofluorescence results showed that the adhesion and F-acti of drug-resistant cells were down regulated after ADAM22. The formation of N fibers was significantly inhibited. At the same time, the ability of the endothelium in vitro and the Mo T culture, the ability of clonformation and the formation of FLPs to decrease the.4. down ADAM22 expression, the adhesion related molecule FAK and the phosphorylation level of Paxillin were significantly inhibited by.GST-pulldown combined Wester. The results of N blot showed that the activity of Rho GTP enzyme Rac1 and Cdc42 in drug-resistant cells decreased significantly after interference of ADAM22 expression. The Western blot results against ITGB1 activation epitope HUTS4 showed that the activity decreased after the ADAM22 expression decreased. Immune coprecipitation results show that ITGB1 can interact with wild type ADAM22 but can not interact with Disintegrin missing mutant ADAM22. Wild type and non mutant ADAM22 can remediate endogenous ADAM22 to inhibit the adhesion of gastric cancer resistant cells, migration and the ability of 3D cloning to inhibit the tolerance to gastric cancer The down-regulation of ADAM22, Rac1 or Cdc42 in the drug cells could significantly reduce the IC50.q RT-PCR and Western blot results of SGC7901/ADR and SGC7901/VCR to a variety of chemotherapeutic drugs. The expression of ADAM22 or Rac1 and Cdc42 in the drug resistant cells of gastric cancer could significantly reduce the expression of alpha and protein and its encoding protein in gastric cancer metastasis. The expression level of the ADAM22 was significantly higher than that of the corresponding primary tissue. The correlation analysis showed that the expression of ADAM22 in gastric cancer tissues was positively correlated with the expression of HUTS4, and the high expression of ADAM22 and HUTS4 in gastric cancer tissues was associated with the.Cox multiple regression analysis of the prognosis of the patients. The high expression of ADAM22 or HUTS4 and the tumor were found to be highly expressed and tumor. N staging and patient age are independent risk predictors of poor prognosis in patients. [Conclusion] this topic screening and clarifying a signal pathway to promote the invasion and metastasis of gastric cancer cells: chemotherapeutic drugs induce high expression of ADAM22 in gastric cancer cells, and the latter increases the adhesion of tumor cells to the extracellular matrix through Disintegrin structure stimulated by ITGB1 The penetration of HIF1 and ABCB1 and the expression of the encoded protein can promote the multidrug resistance of gastric cancer cells. This study deepens the recognition of the drug resistance and metastasis of gastric cancer cells. It also provides a target for developing strategies for gastric cancer resistance and metastasis.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.2
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本文編號(hào)：1863514