本文選題：食管鱗癌 + p38α　； 參考：《新疆醫(yī)科大學》2016年博士論文

【摘要】：目的:本研究旨在探討p38 MAPK4亞基(即p38α,p38β,p38γ和p38δ)在食管鱗癌細胞惡性行為中的作用,探討p38α,p38β,p38γ和p38δ表達的臨床病理學意義;在細胞水平分析p38α,p38β,p38γ和p38δ在細胞增殖,遷移,浸潤,周期和凋亡中的作用;同時,構(gòu)建p38γ和p38δ慢病毒干擾及過表達載體,流式細胞儀分選陽性細胞,皮下注射裸小鼠構(gòu)建皮下荷瘤動物模型,觀察p38γ和p38δ在體內(nèi)成瘤中的作用。方法:運用免疫組織化學技術(shù)在食管鱗癌的組織芯片上檢測p38β,p38γ和p38δ的表達水平,運用卡方統(tǒng)計學分析方法分析p38β,p38γ和p38δ表達的臨床病理學意義(包括患者年齡,性別,臨床分期,T分期,N分期,分化程度,腫瘤大小及大類型);同時,運用Kaplan-Meier生存曲線分析p38β,p38γ和p38δ表達水平與患者的總預(yù)后的統(tǒng)計學相關(guān)性。在體外食管癌細胞系Eca109中,針對p38α,p38β,p38γ和p38δ的cDNA序列,我們分別構(gòu)建了短發(fā)夾RNA干擾載體(shRNA)和真核過表達載體,Lipofectamine 2,000轉(zhuǎn)染Eca109細胞;運用western-blot檢測所構(gòu)建干擾或過表達質(zhì)粒的轉(zhuǎn)染效果;運用細胞劃痕實驗檢測p38α,p38β,p38γ和p38δ干擾或過表達后食管癌細胞Eca109的遷移變化;運用Transwell實驗檢測p38α,p38β,p38γ和p38δ干擾或過表達后食管癌細胞Eca109的浸潤變化;運用流式細胞儀檢測細胞周期和凋亡的變化。最后針對研究相對較少的p38γ和p38δ,我們分別構(gòu)建了其慢病毒干擾和過表達載體,轉(zhuǎn)染Eca109細胞,建立了穩(wěn)定轉(zhuǎn)染p38γ穩(wěn)定敲低和p38δ過表達的Eca109細胞系,并用流式細胞儀進行分選出陽性細胞。最后將流式細胞儀分選出陽性細胞,擴大培養(yǎng),并且皮下注射裸小鼠,構(gòu)建Eca109細胞皮下荷瘤小鼠模型,觀察p38γ和p38δ基因在體內(nèi)對腫瘤細胞增殖的影響。結(jié)果:第一部分:相比癌旁正常組織,p38α在食管癌組織中顯著性高表達(P=0.000);其表達水平與臨床病理學參數(shù),如:年齡(P=0.436),性別(P=1.000),臨床分期(P=0.514),T分期(P=0.429),N分期(P=0.646),分化程度(P=0.670),腫瘤大小(p=0.610)和大體類型(p=0.551)均不具有統(tǒng)計學相關(guān)性,與患者的總預(yù)后也不具有統(tǒng)計學相關(guān)性(p0.05);相比癌旁正常組織,p38β在食管癌組織中顯著性高表達,其差異具有統(tǒng)計學意義(p=0.000);且p38β表達與腫瘤大小具有統(tǒng)計學相關(guān)性(p=0.044)。但與其他臨床病理學參數(shù),如,性別(p=0.730),年齡(p=0.294),臨床分期(p=0.118),t分期(p=0.692),n分期(p=0.109),分化程度(p=0.470)和大體類型(p=0.609)均不具有統(tǒng)計學相關(guān)性;p38γ在癌和癌旁組織中表達不具有統(tǒng)計學相關(guān)性(p=0.365),與性別(p=1.000),年齡(p=0.609),t分期(p=0.063),分化程度(p=0.0.093)和大體類型(p=0.315)等參數(shù)均無統(tǒng)計學相關(guān)性,但是與臨床分期(p=0.017),n分期(p=0.008),腫瘤大小(p=0.017)具有統(tǒng)計學相關(guān)性;相比癌旁正常組織,p38δ在食管癌組織中顯著性高表達,其差異具有統(tǒng)計學意義(p=0.000);但是p38δ的表達與其他臨床病理學參數(shù),如性別(p=1.000),年齡(p=0.603),臨床分期(p=0.089),t分期(p=0.646),n分期(p=0.568),分化程度(p=0.791)和大體類型(p=0.575)均不具有統(tǒng)計學相關(guān)性。運用kaplan-meier生存曲線分析p38β的表達與食管癌患者的總預(yù)后具有統(tǒng)計學相關(guān)性(p=0.012),p38γ和p38δ的表達與患者總預(yù)后均無統(tǒng)計學相關(guān)性(p=0.667和p=0.364)。第二部分:在體外食管癌細胞系水平,我們發(fā)現(xiàn)干擾p38α,p38β,p38γ和p38δ亞基后,相比對照組p38α能夠顯著性地促進eca109的增殖,遷移和浸潤;但是干擾p38β,p38γ和p38δ亞基后能夠顯著性地抑制eca109的增殖,遷移和浸潤。轉(zhuǎn)染真核過表達質(zhì)粒后,表型則相反。流式細胞分析結(jié)果顯示,相比對照組,干擾p38α后能夠抑制能夠顯著性地抑制eca109的凋亡,周期s期顯著性增加;干擾p38β,p38γ和p38δ亞基后能夠抑制能夠顯著性地促進eca109的凋亡,周期s期顯著性降低;轉(zhuǎn)染真核過表達質(zhì)粒后,凋亡和周期表型則相反。第三部分:皮下荷瘤小鼠模型結(jié)果顯示,與對照組相比,p38δ亞基過表達后,在裸鼠皮下能夠顯著性的促進腫瘤生長;與對照組相比,而p38γ敲低后,在裸鼠皮下能夠顯著性的抑制腫瘤生長。提示p38γ和p38δ亞基均能促進體內(nèi)食管癌細胞eca109細胞的增殖。結(jié)論:(1)在臨床組織水平除了p38γ亞基在食管癌和癌旁組織中表達沒有統(tǒng)計學差異外,其余三個亞基,即p38α,p38β和p38δ亞基均在食管癌組織中顯著性高表達。臨床組織水平結(jié)果提示p38γ與食管癌淋巴結(jié)轉(zhuǎn)移相關(guān);p38α,p38β和p38δ亞基與食管癌發(fā)生發(fā)展相關(guān);(2)在體外食管癌細胞系水平,p38α起著抑制食管癌細胞惡性表型的功能;p38β,p38γ和p38δ亞基則起著促進食管癌細胞惡性表型的功能;(3)在體內(nèi)裸鼠動物水平,p38γ和p38δ亞基在體內(nèi)均能夠促進腫瘤生長,起著促癌基因的作用。
[Abstract]:Objective: To investigate the role of p38 MAPK4 subunits (p38, p38 beta, p38 gamma and p38 delta) in malignant behavior of squamous cell carcinoma of the esophagus, to explore the clinicopathological significance of the expression of p38 a, p38 beta, p38 gamma and p38 Delta, and to analyze the role of p38 alpha, p38 beta, gamma and delta in cell proliferation, migration, infiltration, cycle and apoptosis in cell level. 38 gamma and p38 delta lentivirus interference and overexpression vector, flow cytometer sorting positive cells, subcutaneous injection of nude mice to construct subcutaneous tumor bearing animal model, and observe the role of p38 gamma and p38 Delta in tumor formation. Methods: immunohistochemical technique was used to detect the expression level of p38 beta, p38 gamma and p38 Delta on the tissue microarray of esophageal squamous cell carcinoma, and the application card was used. The clinicopathological significance of the expression of p38 beta, p38 gamma and p38 Delta (including patient's age, sex, clinical stage, T staging, N stage, differentiation, tumor size and large type) was analyzed by the method of statistical analysis. At the same time, the Kaplan-Meier survival curve was used to analyze the statistical correlation between the level of p38 beta, p38 gamma and p38 Delta expression and the total prognosis of the patients. In the outer esophageal carcinoma cell line Eca109, for the cDNA sequence of p38 alpha, p38 beta, p38 gamma and p38 Delta, we constructed the short hairpin RNA interference carrier (shRNA) and the eukaryotic overexpression vector, Lipofectamine 2000 transfected Eca109 cells. The transfection effect of the interference or overtable plasmid was constructed by Western-blot detection, and the cell scratch test was used to detect the p38 alpha. P38 beta, p38 gamma and p38 delta interference or overexpressed migration of Eca109 in esophageal cancer cells. Transwell test was used to detect the changes in p38 alpha, p38 beta, p38 gamma and p38 delta interference and the changes in the infiltration of Eca109 in esophageal cancer cells. Flow cytometry was used to detect cell cycle and apoptosis. Finally, we studied relatively less p38 gamma and p38 Delta. The lentivirus interference and overexpression vector were constructed, and the transfected Eca109 cells were transfected to stable transfection of Eca109 cell lines with stable p38 gamma knockout and p38 delta overexpression, and the positive cells were selected by flow cytometry. Finally, the positive cells were selected by flow cytometry to expand culture and subcutaneous injection of nude mice to construct Eca109 cells. The effect of p38 gamma and p38 delta gene on the proliferation of tumor cells in the body was observed in the subcutaneous tumor bearing mice. Results: the first part: the significant high expression of p38 alpha in the esophageal carcinoma tissues (P=0.000) was compared with the normal tissue adjacent to the carcinoma (P=0.000); the expression level and the clinicopathological parameters, such as the age (P=0.436), the sex (P=1.000), the clinical stage (P=0.514), and the T staging (P=0.) 429), N staging (P=0.646), degree of differentiation (P=0.670), tumor size (p=0.610) and gross type (p=0.551) were not statistically related, and had no statistical correlation with the total prognosis of the patients (P0.05). Compared with normal tissues beside cancer, p38 beta was highly expressed in esophageal cancer tissues, and the difference was statistically significant (p=0.000); and p38 beta (p38 beta). The expression was statistically correlated with tumor size (p=0.044), but there was no statistical correlation with other clinicopathological parameters, such as sex (p=0.730), age (p=0.294), clinical staging (p=0.118), T staging (p=0.692), N staging (p=0.109), the degree of differentiation (p=0.470) and general type (p=0.609), and p38 gamma was not expressed in cancer and para cancer tissues. Statistical correlation (p=0.365) was not statistically correlated with sex (p=1.000), age (p=0.609), T staging (p=0.063), degree of differentiation (p=0.0.093) and gross type (p=0.315), but was statistically correlated with the clinical stage (p=0.017), N staging (p=0.008), and tumor size (p=0.017); p38 delta was in the esophagus compared to the normal tissue adjacent to the cancer. The significant high expression in cancer tissues was statistically significant (p=0.000), but the expression of p38 delta was not statistically related to other clinicopathological parameters, such as sex (p=1.000), age (p=0.603), clinical staging (p=0.089), T staging (p=0.646), N staging (p=0.568), differentiation degree (p=0.791) and general type (p=0.575). An-meier survival curve analysis showed that the expression of p38 beta was statistically related to the total prognosis of patients with esophageal cancer (p=0.012). The expression of p38 gamma and p38 delta was not statistically correlated with the total prognosis of patients (p=0.667 and p=0.364). The second part: in vitro esophageal cancer cell line level, we found that the interference of p38 a, p38 beta, p38 gamma, and p38 delta subunits, compared with that of the patients. P38 alpha could significantly promote the proliferation, migration and infiltration of Eca109, but the interference of p38 beta, p38 gamma and p38 delta subunits could significantly inhibit the proliferation, migration and infiltration of Eca109. After transfection of eukaryotic overexpressed plasmids, the phenotype was opposite. The results of flow cytometry showed that the inhibition of p38 alpha could be significantly inhibited compared to the control group. Inhibition of Eca109 apoptosis and periodic S phase increased significantly; interference with p38 beta, p38 gamma and p38 delta subunits could significantly inhibit the apoptosis of Eca109, and the period s phase decreased significantly; after transfection of eukaryotic overexpressed plasmid, apoptosis and cyclical phenotypes were opposite. The third part: subcutaneous tumor bearing mice model results showed p38 delta compared with the control group. Subunits could significantly promote tumor growth in the subcutaneous subcutaneous tissue of nude mice. Compared with the control group, the subunits of p38 gamma could significantly inhibit tumor growth in the nude mice. Suggesting that p38 gamma and p38 delta subunits can promote the proliferation of Eca109 cells in human esophageal cancer cells. (1) at the clinical level, the p38 gamma subunit in the esophagus is in the esophagus. The expression of the other three subunits, p38 a, p38 beta and p38 delta subunits in the esophageal cancer tissues, was significantly higher in cancer and adjacent tissues. The results of clinical tissue showed that p38 gamma was associated with lymph node metastasis of esophageal cancer; p38 alpha, p38 beta and p38 delta subunits were associated with the development of esophageal cancer; (2) in vitro esophageal cancer cell lines Level, p38 alpha plays a role in inhibiting the malignant phenotype of esophageal cancer cells; p38 beta, p38 gamma and p38 delta subunits play a role in promoting malignant phenotype of esophageal cancer cells. (3) in vivo, p38 gamma and p38 delta subunits of p38 gamma and p38 delta subunits can promote tumor growth in vivo and play the role of promoting oncogene.
【學位授予單位】：新疆醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R735.1
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本文編號：2083872