反义葡萄糖转运蛋白1对喉癌Hep-2细胞放射增敏作用及机制的体外研究

发布时间：2018-02-04 22:15

  本文关键词： 葡萄糖转运蛋白-1 HEP-2细胞 喉癌 反义寡核苷酸 放射治疗　出处：《浙江大学》2012年硕士论文　论文类型：学位论文

【摘要】：背景和目的：喉癌是放射治疗效果不佳的常见头颈部恶性肿瘤,通过增强放射治疗效果,避免手术、保留该器官的完整功能是科技工作者面临的难题。放射抵抗往往导致喉功能的丧失或影响患者生命。但放射抵抗的真正机制尚不十分清楚,随着分子生物技术的发展,发现肿瘤内乏氧细胞的存在是肿瘤对射线产生抵抗性的重要原因之一,恶性细胞所处的生理环境能量相对匮乏,呈现局部缺氧状态,并引起肿瘤细胞因子的过度表达。恶性肿瘤细胞的葡萄糖代谢率较高,这种现象已被正电子发射计算机断层(Positron emission tomography, PET)检查所证实。恶性肿瘤细胞对葡萄糖的代谢率增高与葡萄糖转运蛋白(Glucose transporter protein, GLUT)及基因的异常表达有关,其中葡萄糖转运蛋白-1(G LUT-1)在恶性肿瘤细胞的葡萄糖吸收和转运中起主导作用。 国内外研究发现GLUT-1的表达异常与肿瘤的生物学行为有关我们的前期研究已经发现,GLUT-1均异常表达与头颈部癌的生物学行为有关,与头颈部癌的预后差有关,可以作为靶向治疗的新方法之一,并且我们已通过反义寡脱氧核苷酸技术阻断喉癌Hep-2细胞GLUT-1的表达,能抑制喉癌细胞的葡萄糖吸收的影响及Hep-2细胞增殖,可能是喉癌治疗的一种理想靶标。尽管GLUT-1的异常表达不能作为肿瘤放射抵抗的唯一解释,但它在肿瘤放射生物学中的意义已越来越受重视。抑制GLUT-1表达以提高放射敏感性,成为恶性肿瘤靶向治疗的热点之一。在国内外文献中,未见通过靶向抑制GLUT-1的表达提高喉癌的放射敏感性。 本研究采用反义核苷酸技术抑制GLUT-1的表达,阐明GLUT-1表达改变影响喉癌细胞能量供应及增强喉癌细胞对放射敏感性的机制,进一步揭示喉癌细胞对放射抵抗的机理,为喉癌患者功能性治疗和改善预后提供一定的理论基础。 方法： 喉癌Hep-2细胞,转染反义GLUT-1前后,用不同剂量0Gy(对照组)、2Gy、4Gy、8Gy、12Gy的X线照射,照射后继续培养24h、48h、72h后,MTS的方法检测各组Hep-2细胞的存活率,RT-PCR的方法分别检测各组Hep-2细胞GLUT-1mRNA的表达,流式细胞仪检测Hep-2细胞细胞的周期及凋亡 结果： 1转染前,MTS结果显示照射24h后X射线照射没有表现出明显的细胞损伤作用,反而出现了4Gy剂量Hep-2细胞存活率的增高(p0.05)；相同剂量照射随着培养时间的延长,2gy细胞存活率无明显变化(p0.05),4gy和8gy在照射后24h出现存活率增高,之后随着剂量增高和培养时间延长存活率下降；RT-PCR结果显示X线照射组较对照组出现GLUT-1mRNA的表达增高(p0.05)；细胞周期结果显示X线照射后Hep-2细胞出现了G2/M期阻滞；细胞凋亡结果显示随着照射剂量增加及时间的延长,细胞凋亡率增加(p0.05)。 2转染后,MTS结果显示Hep-2细胞,分别在2Gy、4Gy、8Gy、12Gy剂量照射后,随着时间的延长,Hep-2细胞细胞存活率逐渐下降(p0.05)；在同一时间,随着照射剂量增高,细胞存活率降低,差异有显著性；RT-PCR结果显示转染反义GLUT-1前后,反义GLUT-1能抑制Hep-2细胞GLUT-1mRNA的表达。但在放疗后,这种抑制作用不是十分明显；流式细胞仪检测细胞周期发现出现了G2/M期阻滞,但G2/M期阻滞与转染前变化不大(p0.05)；流式细胞仪测细胞凋亡显示转染组均较未转染组细胞凋亡率增高(p0.05),这种转染随着时间延长、照射剂量增大而增高(p0.05)。 结论：喉癌Hep-2细胞对X线照射产生了放射抵抗或者不敏感性,可能与GLUT-1mRNA的表达增高有关；转染反义GLUT-1提高Hep-2细胞对放射的敏感性,可能主要是通过增加细胞凋亡而发挥作用。
[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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