食管鳞癌细胞MMP-9的缺氧应答及在侵袭与转移中的作用

发布时间：2018-06-20 08:11

  本文选题：食管癌 + HIF-1a　； 参考：《郑州大学》2017年硕士论文

【摘要】：研究背景食管癌(esophageal carcinoma,EC)是我国常见的消化系统恶性肿瘤之一。世界范围内,食管癌患者的死亡率在肿瘤患者死亡率中位列第六。近些年来随着医学的发展,食管癌早期诊断及早期治疗积极的开展,其预后虽然有所改善,但由于肿瘤的复发和转移导致患者整个群体的5年生存率依旧不容乐观,肿瘤发生侵袭转移仍是导致患者术后复发和死亡的主要因素。缺氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)和金属基质蛋白酶-9(matrix metalloproteinase,MMP-9)在近年来的研究中被发现与人类多种恶性肿瘤的浸润和转移过程存在着密切的联系。HIF-1α和MMP-9在许多恶性肿瘤中具有呈高表达,并与肿瘤的侵袭转移相关。有研究报道称,在乳腺癌、肺癌细胞中,HIF-1α可通过上调MMP-9的表达促进增强肿瘤细胞的侵袭能力。截至目前,在食管鳞癌中HIF-1α能否通过调控MMP-9的表达进而影响肿瘤细胞的侵袭能力尚未见报道。方法:1.采用免疫组化SP法检测91例食管鳞癌组织和癌旁正常组织中HIF-lα和MMP-9蛋白的表达情况。2.分析HIF-lα与MMP-9的表达关系及二者分别与性别、年龄、肿瘤分化程度、浸润深度、淋巴结转移及肿瘤TNM分期等临床病理参数的关系。3.利用150μmol/L Co Cl2对食管癌EC-1细胞进行0h、12h和24h的刺激时间后,分别采用Real-time PCR和Western blot法对HIF-lα和MMP-9的m RNA和蛋白的表达变化进行检测;采用酶联免疫吸附剂测定法检测细胞上清液中MMP-9蛋白的表达变化。4.HIF-lαsi RNA转染食管癌细胞EC-1细胞后,采用Real-time PCR法和Western blot法分别检测HIF-1a和MMP-9 m RNA和蛋白的表达变化;采用ELISA法检测HIF-lαsi RNA转染后细胞上清液中MMP-9蛋白的表达变化;通过Transwell侵袭小室实验检测HIF-1αsi RNA转染后食管鳞癌EC-1细胞体外侵袭能力的变化。结果:1.91例食管鳞癌组织中,HIF-lα在食管鳞癌组织中的表达(74.725%)较正常食管上皮(39.560%)有明显显著增加,MMP-9的表达食管鳞癌(76.923%)较正常食管上皮(24.175%)的表达有明显显著增加,差异均有统计学意义(P0.05)。2.食管鳞癌组织HIF-1α在不同性别(P0.05)、年龄(P0.05)、组织学分期(P0.05)、浸润深度(P0.05)、有无淋巴结转移(P0.05)中的表达差异无统计学意义(P0.05),而在TNM分期中的表达差异有统计学意义(P0.01)。MMP-9蛋白的表达在不同性别和年龄中的表达无显著性差异(均P0.05),但在不同组织学分级(P0.05)、浸润深度、有无淋巴结转移(P0.01)和TNM分期(P0.05)中的表达有显著性差异。食管鳞癌组织中HIF-lα与MMP-9表达正相关(γs=0.406),差异有统计学意义(P0.05)。3.150μmol/L Co Cl2刺激食管癌EC-1细胞不同时间后,Real-time PCR和Western blot结果显示:与0h相比,Co Cl2刺激12h和24h后,HIF-1αm RNA(1.873±0.231 vs 2.714±0.301 vs 1.000±0.023)和蛋白(1.679±0.104 vs 2.105±0.111 vs0.615±0.092)表达升高,差异有统计学意义(P0.05);与0h相比,Co Cl2刺激12h和24h后,MMP-9 m RNA(1.773±0.255 vs 2.932±0.407 vs 1.000±0.035)和蛋白(1.735±0.255 vs 2.132±0.346 vs 0.754±0.085)表达升高,差异有统计学意义(P0.05)。ELISA结果显示:与0h相比,Co Cl2刺激12h和24h后(632.817±35.335 pg/ml vs 702.141±43.771 vs 386.974±20.260 pg/ml),缺氧后MMP-9的分泌量显著增高(P0.05)。4.4.HIF-1a si RNA转染食管癌细胞EC-1细胞后,Real-time PCR和Western blot结果显示:与常氧组相比,HIF-1αsi RNA转染组HIF-1αm RNA(0.262±0.023vs 1.000±0.048)及蛋白(0.464±0.086 vs 0.694±0.085)表达显著降低,与缺氧组相比,双重作用组HIF-1αm RNA(0.353±0.025 vs 2.351±0.331)和蛋白(0.519±0.154 vs 2.165±0.137)表达显著降低(P0.05);与常氧组相比,HIF-1αsi RNA转染组MMP-9 m RNA(0.315±0.038 vs 1.000±0.052)及蛋白(0.549±0.075 vs 0.774±0.107)表达显著降低,而与缺氧组相比,双重作用组中MMP-9m RNA(0.418±0.044 vs 2.601±0.417)和蛋白(0.623±0.159 vs 2.251±0.133)表达显著降低(P0.05)。Transwell侵袭小室体外侵袭实验结果显示:与常氧组相比,HIF-1αsi RNA转染组(32±6.52 vs 72±7.15)组细胞穿膜数明显降低,与缺氧组相比,双重作用组(45±6.91 vs 156±15.24)的细胞穿膜数显著减少(P0.05)。结论1.在食管鳞癌组织中HIF-1α与MMP-9呈高表达并与肿瘤侵袭转移相关。2.HIF-1α可能通过调控MMP-9的表达进而增强食管鳞癌EC-1细胞的侵袭能力。
[Abstract]:Esophageal carcinoma (EC) is one of the most common digestive malignant tumors in China. The death rate of esophageal cancer patients is sixth in the world. In recent years, with the development of medicine, the early diagnosis and early treatment of esophageal cancer have been actively carried out, but the prognosis has improved, but the prognosis has been improved. The 5 year survival rate of the whole group is still not optimistic in the recurrence and metastasis of the tumor. Tumor invasion and metastasis is still the main factor leading to postoperative recurrence and death. Hypoxia inducible factor -1 alpha (hypoxia inducible factor-1 a, HIF-1 a) and metal matrix protease -9 (matrix metalloproteinase, MMP-9) are in recent years The study has been found to be closely related to the invasion and metastasis of various human malignant tumors..HIF-1 A and MMP-9 are highly expressed in many malignant tumors and are associated with the invasion and metastasis of the tumor. In breast cancer and lung cancer cells, HIF-1 alpha can enhance the expression of MMP-9 to promote the enhancement of tumor cells. Up to now, the expression of HIF-1 alpha in esophageal squamous cell carcinoma has not been reported by regulating the expression of MMP-9 and affecting the invasion ability of tumor cells. Methods: 1. the expression of HIF-l alpha and MMP-9 in 91 cases of esophageal squamous cell carcinoma and normal tissues adjacent to cancer was detected by immunohistochemical SP method. The relationship between the expression of HIF-l A and MMP-9 was analyzed by.2. The relationship between two and two clinical pathological parameters, such as sex, age, tumor differentiation, infiltration depth, lymph node metastasis and tumor TNM staging,.3. using 150 micron Co Cl2 for 0h, 12h, and 24h stimulation time of esophageal cancer EC-1 cells, respectively, to express the expression and protein by Real-time PCR and Western. The changes were detected and the expression of MMP-9 protein in the cell supernatant was detected by enzyme linked immunosorbent assay..4.HIF-l alpha Si RNA was transfected to EC-1 cells of esophageal cancer cells. Real-time PCR method and Western blot method were used to detect the expression of HIF-1a and MMP-9 m and protein expression. Changes in the expression of MMP-9 protein in the supernatant; the invasion ability of EC-1 cells in esophageal squamous cell carcinoma after HIF-1 Si RNA transfection was detected by Transwell invasion. Results: in 1.91 cases of esophageal squamous cell carcinoma, the expression of HIF-l alpha in esophageal squamous cell carcinoma (74.725%) was significantly higher than that of normal esophageal epithelium (39.560%), MMP-9 The expression of esophageal squamous cell carcinoma (76.923%) was significantly higher than that of normal esophageal epithelium (24.175%). The difference was statistically significant (P0.05).2. esophageal squamous cell carcinoma tissue HIF-1 alpha in different sex (P0.05), age (P0.05), histological staging (P0.05), infiltration depth (P0.05), and no lymph node metastasis (P0.05), there was no statistical difference (P0.0) 5), the expression difference in TNM staging was statistically significant (P0.01) expression of.MMP-9 protein had no significant difference in different sex and age (P0.05), but there was a significant difference in the expression of lymph node metastasis (P0.01) and TNM staging (P0.05) in different histological grading (P0.05), P0.01 and TNM stage (P0.05). HIF-l in the squamous cell carcinoma of the esophagus. The positive correlation between alpha and MMP-9 (gamma s=0.406) was significant (P0.05), and the difference was statistically significant (P0.05).3.150 mu mol/L Co Cl2 stimulated esophageal cancer EC-1 cells for different time. .111 vs0.615 + 0.092) increased, and the difference was statistically significant (P0.05). Compared with 0h, Co Cl2 stimulated 12h and 24h, and MMP-9 m RNA (1.773 + 0.255 vs 2.932 + 0.407 1 + 0.035) and protein (1.735 + 0.255 2.132 + 2.132 + 0.754 + 0.085) increased, and the difference was statistically significant After stimulated 12h and 24h (632.817 + 35.335 pg/ml vs 702.141 + 43.771 vs 386.974 + 20.260 pg/ml), the secretion of MMP-9 increased significantly after hypoxia (P0.05).4.4.HIF-1a Si RNA transfection to the cells of esophageal cancer cells. The expression of.048 and protein (0.464 + 0.086 vs 0.694 + 0.085) decreased significantly. Compared with the hypoxia group, the expression of HIF-1 alpha m RNA (0.353 + 0.025 vs 2.351 + 0.331) and protein (0.519 + 0.154 vs 2.165 + 0.137) decreased significantly (P0.05), and the HIF-1 alpha Si RNA transfection group was compared with that of the normal oxygen group. The expression of 49 + 0.075 vs 0.774 + 0.107) decreased significantly. Compared with the anoxia group, the expression of MMP-9m RNA (0.418 + 0.044 vs 2.601 + 0.417) and protein (0.623 + 0.159 vs 2.251 + 0.133) in the double action group decreased significantly (P0.05) in the invasion experiment of.Transwell invasion compartment in vitro, which showed that the HIF-1 a Si RNA transfection group was compared with the normal oxygen group. The number of cell penetrating membranes in the group of + 7.15 decreased significantly. Compared with the anoxia group, the number of cells in the double action group (45 + 6.91 vs 156 + 15.24) decreased significantly (P0.05). Conclusion 1. HIF-1 alpha and MMP-9 were highly expressed in the squamous cell carcinoma of the esophagus and.2.HIF-1 alpha associated with the invasion and metastasis of the tumor may enhance the EC-1 cells of esophageal squamous cell carcinoma by regulating the expression of MMP-9. The ability to attack.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.1
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本文编号：2043612