本文關(guān)鍵詞： 葡萄糖轉(zhuǎn)運(yùn)蛋白-1 HEP-2細(xì)胞 喉癌 反義寡核苷酸 放射治療　出處：《浙江大學(xué)》2012年碩士論文　論文類型：學(xué)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(qiáng)放射治療效果,避免手術(shù)、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(dǎo)致喉功能的喪失或影響患者生命。但放射抵抗的真正機(jī)制尚不十分清楚,隨著分子生物技術(shù)的發(fā)展,發(fā)現(xiàn)腫瘤內(nèi)乏氧細(xì)胞的存在是腫瘤對(duì)射線產(chǎn)生抵抗性的重要原因之一,惡性細(xì)胞所處的生理環(huán)境能量相對(duì)匱乏,呈現(xiàn)局部缺氧狀態(tài),并引起腫瘤細(xì)胞因子的過度表達(dá)。惡性腫瘤細(xì)胞的葡萄糖代謝率較高,這種現(xiàn)象已被正電子發(fā)射計(jì)算機(jī)斷層(Positron emission tomography, PET)檢查所證實(shí)。惡性腫瘤細(xì)胞對(duì)葡萄糖的代謝率增高與葡萄糖轉(zhuǎn)運(yùn)蛋白(Glucose transporter protein, GLUT)及基因的異常表達(dá)有關(guān),其中葡萄糖轉(zhuǎn)運(yùn)蛋白-1(G LUT-1)在惡性腫瘤細(xì)胞的葡萄糖吸收和轉(zhuǎn)運(yùn)中起主導(dǎo)作用。 國(guó)內(nèi)外研究發(fā)現(xiàn)GLUT-1的表達(dá)異常與腫瘤的生物學(xué)行為有關(guān)我們的前期研究已經(jīng)發(fā)現(xiàn),GLUT-1均異常表達(dá)與頭頸部癌的生物學(xué)行為有關(guān),與頭頸部癌的預(yù)后差有關(guān),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(shù)阻斷喉癌Hep-2細(xì)胞GLUT-1的表達(dá),能抑制喉癌細(xì)胞的葡萄糖吸收的影響及Hep-2細(xì)胞增殖,可能是喉癌治療的一種理想靶標(biāo)。盡管GLUT-1的異常表達(dá)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(xué)中的意義已越來(lái)越受重視。抑制GLUT-1表達(dá)以提高放射敏感性,成為惡性腫瘤靶向治療的熱點(diǎn)之一。在國(guó)內(nèi)外文獻(xiàn)中,未見通過靶向抑制GLUT-1的表達(dá)提高喉癌的放射敏感性。 本研究采用反義核苷酸技術(shù)抑制GLUT-1的表達(dá),闡明GLUT-1表達(dá)改變影響喉癌細(xì)胞能量供應(yīng)及增強(qiáng)喉癌細(xì)胞對(duì)放射敏感性的機(jī)制,進(jìn)一步揭示喉癌細(xì)胞對(duì)放射抵抗的機(jī)理,為喉癌患者功能性治療和改善預(yù)后提供一定的理論基礎(chǔ)。 方法： 喉癌Hep-2細(xì)胞,轉(zhuǎn)染反義GLUT-1前后,用不同劑量0Gy(對(duì)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(cè)各組Hep-2細(xì)胞的存活率,RT-PCR的方法分別檢測(cè)各組Hep-2細(xì)胞GLUT-1mRNA的表達(dá),流式細(xì)胞儀檢測(cè)Hep-2細(xì)胞細(xì)胞的周期及凋亡 結(jié)果： 1轉(zhuǎn)染前,MTS結(jié)果顯示照射24h后X射線照射沒有表現(xiàn)出明顯的細(xì)胞損傷作用,反而出現(xiàn)了4Gy劑量Hep-2細(xì)胞存活率的增高(p0.05)；相同劑量照射隨著培養(yǎng)時(shí)間的延長(zhǎng),2gy細(xì)胞存活率無(wú)明顯變化(p0.05),4gy和8gy在照射后24h出現(xiàn)存活率增高,之后隨著劑量增高和培養(yǎng)時(shí)間延長(zhǎng)存活率下降；RT-PCR結(jié)果顯示X線照射組較對(duì)照組出現(xiàn)GLUT-1mRNA的表達(dá)增高(p0.05)；細(xì)胞周期結(jié)果顯示X線照射后Hep-2細(xì)胞出現(xiàn)了G2/M期阻滯；細(xì)胞凋亡結(jié)果顯示隨著照射劑量增加及時(shí)間的延長(zhǎng),細(xì)胞凋亡率增加(p0.05)。 2轉(zhuǎn)染后,MTS結(jié)果顯示Hep-2細(xì)胞,分別在2Gy、4Gy、8Gy、12Gy劑量照射后,隨著時(shí)間的延長(zhǎng),Hep-2細(xì)胞細(xì)胞存活率逐漸下降(p0.05)；在同一時(shí)間,隨著照射劑量增高,細(xì)胞存活率降低,差異有顯著性；RT-PCR結(jié)果顯示轉(zhuǎn)染反義GLUT-1前后,反義GLUT-1能抑制Hep-2細(xì)胞GLUT-1mRNA的表達(dá)。但在放療后,這種抑制作用不是十分明顯；流式細(xì)胞儀檢測(cè)細(xì)胞周期發(fā)現(xiàn)出現(xiàn)了G2/M期阻滯,但G2/M期阻滯與轉(zhuǎn)染前變化不大(p0.05)；流式細(xì)胞儀測(cè)細(xì)胞凋亡顯示轉(zhuǎn)染組均較未轉(zhuǎn)染組細(xì)胞凋亡率增高(p0.05),這種轉(zhuǎn)染隨著時(shí)間延長(zhǎng)、照射劑量增大而增高(p0.05)。 結(jié)論：喉癌Hep-2細(xì)胞對(duì)X線照射產(chǎn)生了放射抵抗或者不敏感性,可能與GLUT-1mRNA的表達(dá)增高有關(guān)；轉(zhuǎn)染反義GLUT-1提高Hep-2細(xì)胞對(duì)放射的敏感性,可能主要是通過增加細(xì)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.
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本文編號(hào)：1491294