本文關鍵詞： 葡萄糖轉運蛋白-1 HEP-2細胞 喉癌 反義寡核苷酸 放射治療　出處：《浙江大學》2012年碩士論文　論文類型：學位論文

【摘要】：背景和目的：喉癌是放射治療效果不佳的常見頭頸部惡性腫瘤,通過增強放射治療效果,避免手術、保留該器官的完整功能是科技工作者面臨的難題。放射抵抗往往導致喉功能的喪失或影響患者生命。但放射抵抗的真正機制尚不十分清楚,隨著分子生物技術的發(fā)展,發(fā)現腫瘤內乏氧細胞的存在是腫瘤對射線產生抵抗性的重要原因之一,惡性細胞所處的生理環(huán)境能量相對匱乏,呈現局部缺氧狀態(tài),并引起腫瘤細胞因子的過度表達。惡性腫瘤細胞的葡萄糖代謝率較高,這種現象已被正電子發(fā)射計算機斷層(Positron emission tomography, PET)檢查所證實。惡性腫瘤細胞對葡萄糖的代謝率增高與葡萄糖轉運蛋白(Glucose transporter protein, GLUT)及基因的異常表達有關,其中葡萄糖轉運蛋白-1(G LUT-1)在惡性腫瘤細胞的葡萄糖吸收和轉運中起主導作用。 國內外研究發(fā)現GLUT-1的表達異常與腫瘤的生物學行為有關我們的前期研究已經發(fā)現,GLUT-1均異常表達與頭頸部癌的生物學行為有關,與頭頸部癌的預后差有關,可以作為靶向治療的新方法之一,并且我們已通過反義寡脫氧核苷酸技術阻斷喉癌Hep-2細胞GLUT-1的表達,能抑制喉癌細胞的葡萄糖吸收的影響及Hep-2細胞增殖,可能是喉癌治療的一種理想靶標。盡管GLUT-1的異常表達不能作為腫瘤放射抵抗的唯一解釋,但它在腫瘤放射生物學中的意義已越來越受重視。抑制GLUT-1表達以提高放射敏感性,成為惡性腫瘤靶向治療的熱點之一。在國內外文獻中,未見通過靶向抑制GLUT-1的表達提高喉癌的放射敏感性。 本研究采用反義核苷酸技術抑制GLUT-1的表達,闡明GLUT-1表達改變影響喉癌細胞能量供應及增強喉癌細胞對放射敏感性的機制,進一步揭示喉癌細胞對放射抵抗的機理,為喉癌患者功能性治療和改善預后提供一定的理論基礎。 方法： 喉癌Hep-2細胞,轉染反義GLUT-1前后,用不同劑量0Gy(對照組)、2Gy、4Gy、8Gy、12Gy的X線照射,照射后繼續(xù)培養(yǎng)24h、48h、72h后,MTS的方法檢測各組Hep-2細胞的存活率,RT-PCR的方法分別檢測各組Hep-2細胞GLUT-1mRNA的表達,流式細胞儀檢測Hep-2細胞細胞的周期及凋亡 結果： 1轉染前,MTS結果顯示照射24h后X射線照射沒有表現出明顯的細胞損傷作用,反而出現了4Gy劑量Hep-2細胞存活率的增高(p0.05)；相同劑量照射隨著培養(yǎng)時間的延長,2gy細胞存活率無明顯變化(p0.05),4gy和8gy在照射后24h出現存活率增高,之后隨著劑量增高和培養(yǎng)時間延長存活率下降；RT-PCR結果顯示X線照射組較對照組出現GLUT-1mRNA的表達增高(p0.05)；細胞周期結果顯示X線照射后Hep-2細胞出現了G2/M期阻滯；細胞凋亡結果顯示隨著照射劑量增加及時間的延長,細胞凋亡率增加(p0.05)。 2轉染后,MTS結果顯示Hep-2細胞,分別在2Gy、4Gy、8Gy、12Gy劑量照射后,隨著時間的延長,Hep-2細胞細胞存活率逐漸下降(p0.05)；在同一時間,隨著照射劑量增高,細胞存活率降低,差異有顯著性；RT-PCR結果顯示轉染反義GLUT-1前后,反義GLUT-1能抑制Hep-2細胞GLUT-1mRNA的表達。但在放療后,這種抑制作用不是十分明顯；流式細胞儀檢測細胞周期發(fā)現出現了G2/M期阻滯,但G2/M期阻滯與轉染前變化不大(p0.05)；流式細胞儀測細胞凋亡顯示轉染組均較未轉染組細胞凋亡率增高(p0.05),這種轉染隨著時間延長、照射劑量增大而增高(p0.05)。 結論：喉癌Hep-2細胞對X線照射產生了放射抵抗或者不敏感性,可能與GLUT-1mRNA的表達增高有關；轉染反義GLUT-1提高Hep-2細胞對放射的敏感性,可能主要是通過增加細胞凋亡而發(fā)揮作用。
[Abstract]:Background and objective: laryngeal carcinoma is a common malignant tumor in head and neck radiotherapy effect, avoid surgery by enhancing the therapeutic effect, radiation, retaining the integrity function of this organ is facing the problem of science and technology workers. Radiation resistance often leads to loss of laryngeal function or affect the lives of patients. But the real mechanism of radiation resistance is not very clear, with the the development of molecular biology, found that hypoxic cells within the tumor is one of the important reasons for tumor resistant to radiation, the malignant cells in the physiological environment of energy scarcity, showing local hypoxia, and excessive expression of tumor cell factor of malignant tumor cells. The glucose metabolism rate is higher, this phenomenon has been positron emission computed tomography (Positron emission tomography, PET) confirmed by examination. The metabolism of malignant tumor cells to glucose and fructose increased Glucose transporter (Glucose transporter, protein, GLUT) and abnormal expression of genes, including glucose transporter -1 (G LUT-1) play a leading role in malignant tumor cell glucose uptake and transport.
The studies showed that the expression of GLUT-1 and tumor related abnormal biological behavior of our previous studies have found that abnormal expression of GLUT-1 were related with biological behavior of head and neck cancer, and prognosis of head and neck cancer is poor, can be used as the target of a new approach to the treatment, and we have been through blocking the expression of GLUT-1 in human laryngeal carcinoma Hep-2 cells antisense oligodeoxynucleotides can inhibit the proliferation of HEp-2 cells, glucose uptake and Hep-2 cells may be an ideal target for the treatment of laryngeal cancer. Despite the abnormal expression of GLUT-1 is not the only explanation of radiation resistance of tumor radiotherapy and its significance in tumor, but in radiation biology has attracted more and more attention. The suppression of GLUT-1 expression in order to improve the radiosensitivity of malignant tumor targeting, become one of the hot treatment. In the domestic and foreign literature, not to improve laryngeal cancer by inhibiting the expression of GLUT-1 target Radiosensitivity.
The expression of antisense oligodeoxynucleotide inhibits GLUT-1, clarify the changes of expression of GLUT-1 in laryngeal carcinoma cells and enhance the effect of energy supply mechanism of laryngeal carcinoma cells on radiosensitivity, further reveal the mechanism of radiation resistance of laryngeal squamous cell carcinoma, to provide a theoretical basis for the function of patients with laryngeal cancer treatment and improving the prognosis.
Method錛，

本文編號：1491294