本文選題：食管癌 + HIF-1a　； 參考：《鄭州大學》2017年碩士論文

【摘要】：研究背景食管癌(esophageal carcinoma,EC)是我國常見的消化系統(tǒng)惡性腫瘤之一。世界范圍內(nèi),食管癌患者的死亡率在腫瘤患者死亡率中位列第六。近些年來隨著醫(yī)學的發(fā)展,食管癌早期診斷及早期治療積極的開展,其預(yù)后雖然有所改善,但由于腫瘤的復發(fā)和轉(zhuǎn)移導致患者整個群體的5年生存率依舊不容樂觀,腫瘤發(fā)生侵襲轉(zhuǎn)移仍是導致患者術(shù)后復發(fā)和死亡的主要因素。缺氧誘導因子-1α(hypoxia inducible factor-1α,HIF-1α)和金屬基質(zhì)蛋白酶-9(matrix metalloproteinase,MMP-9)在近年來的研究中被發(fā)現(xiàn)與人類多種惡性腫瘤的浸潤和轉(zhuǎn)移過程存在著密切的聯(lián)系。HIF-1α和MMP-9在許多惡性腫瘤中具有呈高表達,并與腫瘤的侵襲轉(zhuǎn)移相關(guān)。有研究報道稱,在乳腺癌、肺癌細胞中,HIF-1α可通過上調(diào)MMP-9的表達促進增強腫瘤細胞的侵襲能力。截至目前,在食管鱗癌中HIF-1α能否通過調(diào)控MMP-9的表達進而影響腫瘤細胞的侵襲能力尚未見報道。方法:1.采用免疫組化SP法檢測91例食管鱗癌組織和癌旁正常組織中HIF-lα和MMP-9蛋白的表達情況。2.分析HIF-lα與MMP-9的表達關(guān)系及二者分別與性別、年齡、腫瘤分化程度、浸潤深度、淋巴結(jié)轉(zhuǎn)移及腫瘤TNM分期等臨床病理參數(shù)的關(guān)系。3.利用150μmol/L Co Cl2對食管癌EC-1細胞進行0h、12h和24h的刺激時間后,分別采用Real-time PCR和Western blot法對HIF-lα和MMP-9的m RNA和蛋白的表達變化進行檢測;采用酶聯(lián)免疫吸附劑測定法檢測細胞上清液中MMP-9蛋白的表達變化。4.HIF-lαsi RNA轉(zhuǎn)染食管癌細胞EC-1細胞后,采用Real-time PCR法和Western blot法分別檢測HIF-1a和MMP-9 m RNA和蛋白的表達變化;采用ELISA法檢測HIF-lαsi RNA轉(zhuǎn)染后細胞上清液中MMP-9蛋白的表達變化;通過Transwell侵襲小室實驗檢測HIF-1αsi RNA轉(zhuǎn)染后食管鱗癌EC-1細胞體外侵襲能力的變化。結(jié)果:1.91例食管鱗癌組織中,HIF-lα在食管鱗癌組織中的表達(74.725%)較正常食管上皮(39.560%)有明顯顯著增加,MMP-9的表達食管鱗癌(76.923%)較正常食管上皮(24.175%)的表達有明顯顯著增加,差異均有統(tǒng)計學意義(P0.05)。2.食管鱗癌組織HIF-1α在不同性別(P0.05)、年齡(P0.05)、組織學分期(P0.05)、浸潤深度(P0.05)、有無淋巴結(jié)轉(zhuǎn)移(P0.05)中的表達差異無統(tǒng)計學意義(P0.05),而在TNM分期中的表達差異有統(tǒng)計學意義(P0.01)。MMP-9蛋白的表達在不同性別和年齡中的表達無顯著性差異(均P0.05),但在不同組織學分級(P0.05)、浸潤深度、有無淋巴結(jié)轉(zhuǎn)移(P0.01)和TNM分期(P0.05)中的表達有顯著性差異。食管鱗癌組織中HIF-lα與MMP-9表達正相關(guān)(γs=0.406),差異有統(tǒng)計學意義(P0.05)。3.150μmol/L Co Cl2刺激食管癌EC-1細胞不同時間后,Real-time PCR和Western blot結(jié)果顯示:與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)表達升高,差異有統(tǒng)計學意義(P0.05);與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)表達升高,差異有統(tǒng)計學意義(P0.05)。ELISA結(jié)果顯示:與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的分泌量顯著增高(P0.05)。4.4.HIF-1a si RNA轉(zhuǎn)染食管癌細胞EC-1細胞后,Real-time PCR和Western blot結(jié)果顯示:與常氧組相比,HIF-1αsi RNA轉(zhuǎn)染組HIF-1αm RNA(0.262±0.023vs 1.000±0.048)及蛋白(0.464±0.086 vs 0.694±0.085)表達顯著降低,與缺氧組相比,雙重作用組HIF-1αm RNA(0.353±0.025 vs 2.351±0.331)和蛋白(0.519±0.154 vs 2.165±0.137)表達顯著降低(P0.05);與常氧組相比,HIF-1αsi RNA轉(zhuǎn)染組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)表達顯著降低,而與缺氧組相比,雙重作用組中MMP-9m RNA(0.418±0.044 vs 2.601±0.417)和蛋白(0.623±0.159 vs 2.251±0.133)表達顯著降低(P0.05)。Transwell侵襲小室體外侵襲實驗結(jié)果顯示:與常氧組相比,HIF-1αsi RNA轉(zhuǎn)染組(32±6.52 vs 72±7.15)組細胞穿膜數(shù)明顯降低,與缺氧組相比,雙重作用組(45±6.91 vs 156±15.24)的細胞穿膜數(shù)顯著減少(P0.05)。結(jié)論1.在食管鱗癌組織中HIF-1α與MMP-9呈高表達并與腫瘤侵襲轉(zhuǎn)移相關(guān)。2.HIF-1α可能通過調(diào)控MMP-9的表達進而增強食管鱗癌EC-1細胞的侵襲能力。
[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.
【學位授予單位】：鄭州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R735.1
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本文編號：2043612