缺氧微环境影响骨肉瘤放疗抵抗的机制研究
发布时间：2019-06-20 00:26
【摘要】：第一部分缺氧微环境对骨肉瘤细胞放疗抵抗能力的影响目的:研究缺氧微环境对骨肉瘤细胞放疗抵抗能力的影响。方法:分别培养人骨肉瘤MG-63细胞和经过低氧条件预处理的人骨肉瘤MG-63细胞,用梯度剂量X-ray进行照射处理。采用光镜下观察细胞形态,流式细胞技术(FCM)检测各组细胞凋亡,MTT比色法测定细胞增殖率,对比各组细胞增殖及凋亡水平。结果:在常氧条件下,6Gy的辐射剂量可显著抑制MG-63细胞的增殖,给予MG-63细胞缺氧预处理后,辐射损伤对于细胞增殖的抑制作用被显著减弱;6Gy的辐射剂量可显著促进MG-63细胞的凋亡水平增加,给予MG-63细胞缺氧预处理后,辐射损伤对于细胞凋亡水平的促进作用被显著减弱。结论:6Gy可有效抑制骨肉瘤MG-63细胞的增殖,增加细胞凋亡。缺氧微环境显著升高骨肉瘤MG-63细胞增殖、凋亡率降低。缺氧微环境可能在增强骨肉瘤细胞放疗抵抗中发挥重要作用。第二部分缺氧微环境诱导骨肉瘤细胞放疗抵抗的机制目的:研究缺氧微环境诱导骨肉瘤细胞放疗抵抗的具体机制,并对相关机制进行探讨。方法:分别培养人骨肉瘤MG-63细胞和经过低氧条件预处理的人骨肉瘤MG-63细胞。采用重组腺病毒体外转染,Western Blot方法,细胞免疫荧光技术检测细胞DNA损伤(γ-H2AX),MitoSOX染色检测线粒体ROS,检测线粒体膜电位(MMP),流式细胞技术,MTT细胞增殖实验,3-MA抑制自噬实验、凋亡和自噬水平。结果:缺氧预处理可产生以下效应:1)诱导MG-63细胞中自噬水平的升高;2)通过降低细胞辐射损伤密切相关活性氧(ROS)水平达从而降低DNA损伤;3)降低线粒体ROS的产生进而保护了线粒体功能;4)利用自噬抑制剂抑制骨肉瘤细胞自噬后,缺氧微环境对骨肉瘤细胞耐受辐射损伤的保护作用消失。结论:缺氧微环境通过诱导骨肉瘤细胞发生自噬,提高了骨肉瘤细胞耐受辐射损伤的能力,因而导致骨肉瘤细胞发生放疗抵抗。自噬可能介导了缺氧微环境对骨肉瘤细胞耐受辐射损伤的保护作用。第三部分缺氧诱导因子表达与骨肉瘤患者预后的相关性分析目的:研究缺氧诱导因子HIF-1、LC3-II及p62在人骨肉瘤组织中的表达,分析其与临床病理的关系,并探讨HIF-1在骨肉瘤发生和发展中的作用和意义。方法:采用免疫组织化学染色方法检测89例(平均年龄为22岁)骨肉瘤组织中HIF-1的表达水平,并在只接受手术和放疗的患者骨肉瘤标本中进一步检测了LC3-II及p62的表达水平。Kaplan-Meier分析比较HIF-1、LC3-II及p62高表达、中表达和低表达患者生存率之间的差异。结果:HIF-1在骨肉瘤组织中低中高表达比例分别为20.2%(18/89)、24.7%(22/89)、55.1%(49/89),且HIF-1的表达与Enneking分期(P0.01)及远处转移(P0.01)显著相关。而与其他临床病理特征之间无明显的关联。LC3-Ⅱ在36例术后只接受放疗的骨肉瘤患者中低中高表达比例分别为22.2%(8/36)、25%(9/36)、52.8%(19/36)。与此同时,52.8%(19/36)的骨肉瘤组织P62的表达水平较低,25%(9/36)中度表达,22.2%(8/36)高表达。且LC3-Ⅱ及P62的表达与Enneking分期(P0.01),远处转移(P0.01)及患者生存期显著相关。而与其他临床病理特征之间无明显的关联。结论:HIF-1、LC3-Ⅱ在骨肉瘤组织中高表达,而P62低表达。HIF-1、LC3-Ⅱ高表达,P62低表达提示临床预后较差。骨肉瘤患者组织中自噬水平的高低与放疗预后相关。
[Abstract]:The effect of the first part of the hypoxic microenvironment on the radiation resistance of osteosarcoma cells is to study the effect of the hypoxia microenvironment on the radiation resistance of osteosarcoma cells. Methods: The MG-63 cells of human osteosarcoma MG-63 and the human osteosarcoma MG-63 cells pretreated by hypoxia were cultured, and the irradiation treatment was carried out with a gradient dose of X-ray. The cell morphology and flow cytometry (FCM) were used to detect the apoptosis of each group, and the rate of cell proliferation was determined by the MTT method, and the cell proliferation and apoptosis were compared. Results: Under the condition of normal oxygen, the radiation dose of 6 Gy could significantly inhibit the proliferation of MG-63 cells, and the inhibitory effect of radiation injury on cell proliferation was significantly reduced after hypoxic preconditioning of MG-63 cells, and the radiation dose of 6 Gy could significantly increase the apoptosis level of MG-63 cells. The effect of radiation injury on the level of apoptosis of MG-63 cells was significantly reduced after hypoxic preconditioning of MG-63 cells. Conclusion: 6Gy can effectively inhibit the proliferation of MG-63 cells and increase the cell apoptosis. The hypoxia microenvironment significantly increased the proliferation and apoptosis rate of the MG-63 cells. The hypoxic microenvironment may play an important role in the enhancement of the radiation resistance of osteosarcoma cells. The purpose of the second part is to study the mechanism of the radiation resistance of osteosarcoma cells induced by hypoxia microenvironment. Methods: The MG-63 cells of human osteosarcoma MG-63 and the human osteosarcoma MG-63 cells pretreated by hypoxia were cultured. Using recombinant adenovirus in vitro transfection, Western Blot method, cell immunofluorescence technique to detect cell DNA damage (HCO3-H2AX), Mitsox staining to detect mitochondrial ROS, detect mitochondrial membrane potential (MMP), flow cytometry, MTT cell proliferation experiment,3-MA inhibition autophagy experiment, Apoptosis and autophagy. Results: The following effects can be produced by the pre-treatment of hypoxia:1) the level of autophagy in the MG-63 cells is induced;2) the level of reactive oxygen (ROS) related to the radiation damage of the cells is reduced, so that the DNA damage is reduced;3) the generation of the mitochondrial ROS is reduced, and the function of the mitochondria is protected; 4) After the autophagy of the osteosarcoma cells was inhibited by autophagy, the protective effect of the hypoxic microenvironment on the radiation damage of the osteosarcoma cells was disappeared. Conclusion: The hypoxia microenvironment can induce the autophagy of the osteosarcoma cell and increase the ability of the osteosarcoma cell to resist the radiation damage, thus leading to the radiotherapy resistance of the osteosarcoma cell. Autophagy may mediate the protective effect of hypoxia microenvironment on the radiation damage of osteosarcoma cells. Objective: To study the relationship between the expression of hypoxia-inducing factor (HIF-1), LC3-II (LC3-II) and p62 (p62) in human osteosarcoma, and to study the relationship between the expression of HIF-1, LC3-II and p62 in human osteosarcoma, and to explore the role and significance of HIF-1 in the development and development of osteosarcoma. Methods: The expression level of HIF-1 in 89 cases of osteosarcoma (mean age was 22 years) was detected by immunohistochemical staining method, and the expression level of LC3-II and p62 was further detected in osteosarcoma specimens with only operation and radiotherapy. Kaplan-Meier analysis compared the differences between the HIF-1, LC3-II, and p62 high-expression, medium-and low-expression patient survival rates. Results: The expression of HIF-1 in osteosarcoma tissue was 20.2% (18/89), 24.7% (22/89), 55.1% (49/89), and the expression of HIF-1 was significantly correlated with Enneking stage (P0.01) and distant metastasis (P0.01). And there is no significant association with other clinical pathological features. The expression of LC3-鈪，
本文编号：2502758
[Abstract]:The effect of the first part of the hypoxic microenvironment on the radiation resistance of osteosarcoma cells is to study the effect of the hypoxia microenvironment on the radiation resistance of osteosarcoma cells. Methods: The MG-63 cells of human osteosarcoma MG-63 and the human osteosarcoma MG-63 cells pretreated by hypoxia were cultured, and the irradiation treatment was carried out with a gradient dose of X-ray. The cell morphology and flow cytometry (FCM) were used to detect the apoptosis of each group, and the rate of cell proliferation was determined by the MTT method, and the cell proliferation and apoptosis were compared. Results: Under the condition of normal oxygen, the radiation dose of 6 Gy could significantly inhibit the proliferation of MG-63 cells, and the inhibitory effect of radiation injury on cell proliferation was significantly reduced after hypoxic preconditioning of MG-63 cells, and the radiation dose of 6 Gy could significantly increase the apoptosis level of MG-63 cells. The effect of radiation injury on the level of apoptosis of MG-63 cells was significantly reduced after hypoxic preconditioning of MG-63 cells. Conclusion: 6Gy can effectively inhibit the proliferation of MG-63 cells and increase the cell apoptosis. The hypoxia microenvironment significantly increased the proliferation and apoptosis rate of the MG-63 cells. The hypoxic microenvironment may play an important role in the enhancement of the radiation resistance of osteosarcoma cells. The purpose of the second part is to study the mechanism of the radiation resistance of osteosarcoma cells induced by hypoxia microenvironment. Methods: The MG-63 cells of human osteosarcoma MG-63 and the human osteosarcoma MG-63 cells pretreated by hypoxia were cultured. Using recombinant adenovirus in vitro transfection, Western Blot method, cell immunofluorescence technique to detect cell DNA damage (HCO3-H2AX), Mitsox staining to detect mitochondrial ROS, detect mitochondrial membrane potential (MMP), flow cytometry, MTT cell proliferation experiment,3-MA inhibition autophagy experiment, Apoptosis and autophagy. Results: The following effects can be produced by the pre-treatment of hypoxia:1) the level of autophagy in the MG-63 cells is induced;2) the level of reactive oxygen (ROS) related to the radiation damage of the cells is reduced, so that the DNA damage is reduced;3) the generation of the mitochondrial ROS is reduced, and the function of the mitochondria is protected; 4) After the autophagy of the osteosarcoma cells was inhibited by autophagy, the protective effect of the hypoxic microenvironment on the radiation damage of the osteosarcoma cells was disappeared. Conclusion: The hypoxia microenvironment can induce the autophagy of the osteosarcoma cell and increase the ability of the osteosarcoma cell to resist the radiation damage, thus leading to the radiotherapy resistance of the osteosarcoma cell. Autophagy may mediate the protective effect of hypoxia microenvironment on the radiation damage of osteosarcoma cells. Objective: To study the relationship between the expression of hypoxia-inducing factor (HIF-1), LC3-II (LC3-II) and p62 (p62) in human osteosarcoma, and to study the relationship between the expression of HIF-1, LC3-II and p62 in human osteosarcoma, and to explore the role and significance of HIF-1 in the development and development of osteosarcoma. Methods: The expression level of HIF-1 in 89 cases of osteosarcoma (mean age was 22 years) was detected by immunohistochemical staining method, and the expression level of LC3-II and p62 was further detected in osteosarcoma specimens with only operation and radiotherapy. Kaplan-Meier analysis compared the differences between the HIF-1, LC3-II, and p62 high-expression, medium-and low-expression patient survival rates. Results: The expression of HIF-1 in osteosarcoma tissue was 20.2% (18/89), 24.7% (22/89), 55.1% (49/89), and the expression of HIF-1 was significantly correlated with Enneking stage (P0.01) and distant metastasis (P0.01). And there is no significant association with other clinical pathological features. The expression of LC3-鈪，
本文编号：2502758
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