p38MAPK四亚基在食管鳞癌恶性表型中的功能研究

发布时间：2018-06-29 23:40

  本文选题：食管鳞癌 + p38α　； 参考：《新疆医科大学》2016年博士论文

【摘要】：目的:本研究旨在探讨p38 MAPK4亚基(即p38α,p38β,p38γ和p38δ)在食管鳞癌细胞恶性行为中的作用,探讨p38α,p38β,p38γ和p38δ表达的临床病理学意义;在细胞水平分析p38α,p38β,p38γ和p38δ在细胞增殖,迁移,浸润,周期和凋亡中的作用;同时,构建p38γ和p38δ慢病毒干扰及过表达载体,流式细胞仪分选阳性细胞,皮下注射裸小鼠构建皮下荷瘤动物模型,观察p38γ和p38δ在体内成瘤中的作用。方法:运用免疫组织化学技术在食管鳞癌的组织芯片上检测p38β,p38γ和p38δ的表达水平,运用卡方统计学分析方法分析p38β,p38γ和p38δ表达的临床病理学意义(包括患者年龄,性别,临床分期,T分期,N分期,分化程度,肿瘤大小及大类型);同时,运用Kaplan-Meier生存曲线分析p38β,p38γ和p38δ表达水平与患者的总预后的统计学相关性。在体外食管癌细胞系Eca109中,针对p38α,p38β,p38γ和p38δ的cDNA序列,我们分别构建了短发夹RNA干扰载体(shRNA)和真核过表达载体,Lipofectamine 2,000转染Eca109细胞;运用western-blot检测所构建干扰或过表达质粒的转染效果;运用细胞划痕实验检测p38α,p38β,p38γ和p38δ干扰或过表达后食管癌细胞Eca109的迁移变化;运用Transwell实验检测p38α,p38β,p38γ和p38δ干扰或过表达后食管癌细胞Eca109的浸润变化;运用流式细胞仪检测细胞周期和凋亡的变化。最后针对研究相对较少的p38γ和p38δ,我们分别构建了其慢病毒干扰和过表达载体,转染Eca109细胞,建立了稳定转染p38γ稳定敲低和p38δ过表达的Eca109细胞系,并用流式细胞仪进行分选出阳性细胞。最后将流式细胞仪分选出阳性细胞,扩大培养,并且皮下注射裸小鼠,构建Eca109细胞皮下荷瘤小鼠模型,观察p38γ和p38δ基因在体内对肿瘤细胞增殖的影响。结果:第一部分:相比癌旁正常组织,p38α在食管癌组织中显著性高表达(P=0.000);其表达水平与临床病理学参数,如:年龄(P=0.436),性别(P=1.000),临床分期(P=0.514),T分期(P=0.429),N分期(P=0.646),分化程度(P=0.670),肿瘤大小(p=0.610)和大体类型(p=0.551)均不具有统计学相关性,与患者的总预后也不具有统计学相关性(p0.05);相比癌旁正常组织,p38β在食管癌组织中显著性高表达,其差异具有统计学意义(p=0.000);且p38β表达与肿瘤大小具有统计学相关性(p=0.044)。但与其他临床病理学参数,如,性别(p=0.730),年龄(p=0.294),临床分期(p=0.118),t分期(p=0.692),n分期(p=0.109),分化程度(p=0.470)和大体类型(p=0.609)均不具有统计学相关性;p38γ在癌和癌旁组织中表达不具有统计学相关性(p=0.365),与性别(p=1.000),年龄(p=0.609),t分期(p=0.063),分化程度(p=0.0.093)和大体类型(p=0.315)等参数均无统计学相关性,但是与临床分期(p=0.017),n分期(p=0.008),肿瘤大小(p=0.017)具有统计学相关性;相比癌旁正常组织,p38δ在食管癌组织中显著性高表达,其差异具有统计学意义(p=0.000);但是p38δ的表达与其他临床病理学参数,如性别(p=1.000),年龄(p=0.603),临床分期(p=0.089),t分期(p=0.646),n分期(p=0.568),分化程度(p=0.791)和大体类型(p=0.575)均不具有统计学相关性。运用kaplan-meier生存曲线分析p38β的表达与食管癌患者的总预后具有统计学相关性(p=0.012),p38γ和p38δ的表达与患者总预后均无统计学相关性(p=0.667和p=0.364)。第二部分:在体外食管癌细胞系水平,我们发现干扰p38α,p38β,p38γ和p38δ亚基后,相比对照组p38α能够显著性地促进eca109的增殖,迁移和浸润;但是干扰p38β,p38γ和p38δ亚基后能够显著性地抑制eca109的增殖,迁移和浸润。转染真核过表达质粒后,表型则相反。流式细胞分析结果显示,相比对照组,干扰p38α后能够抑制能够显著性地抑制eca109的凋亡,周期s期显著性增加;干扰p38β,p38γ和p38δ亚基后能够抑制能够显著性地促进eca109的凋亡,周期s期显著性降低;转染真核过表达质粒后,凋亡和周期表型则相反。第三部分:皮下荷瘤小鼠模型结果显示,与对照组相比,p38δ亚基过表达后,在裸鼠皮下能够显著性的促进肿瘤生长;与对照组相比,而p38γ敲低后,在裸鼠皮下能够显著性的抑制肿瘤生长。提示p38γ和p38δ亚基均能促进体内食管癌细胞eca109细胞的增殖。结论:(1)在临床组织水平除了p38γ亚基在食管癌和癌旁组织中表达没有统计学差异外,其余三个亚基,即p38α,p38β和p38δ亚基均在食管癌组织中显著性高表达。临床组织水平结果提示p38γ与食管癌淋巴结转移相关;p38α,p38β和p38δ亚基与食管癌发生发展相关;(2)在体外食管癌细胞系水平,p38α起着抑制食管癌细胞恶性表型的功能;p38β,p38γ和p38δ亚基则起着促进食管癌细胞恶性表型的功能;(3)在体内裸鼠动物水平,p38γ和p38δ亚基在体内均能够促进肿瘤生长,起着促癌基因的作用。
[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.
【学位授予单位】：新疆医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.1
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