【摘要】：第一部分缺氧微環(huán)境對骨肉瘤細胞放療抵抗能力的影響目的:研究缺氧微環(huán)境對骨肉瘤細胞放療抵抗能力的影響。方法:分別培養(yǎng)人骨肉瘤MG-63細胞和經過低氧條件預處理的人骨肉瘤MG-63細胞,用梯度劑量X-ray進行照射處理。采用光鏡下觀察細胞形態(tài),流式細胞技術(FCM)檢測各組細胞凋亡,MTT比色法測定細胞增殖率,對比各組細胞增殖及凋亡水平。結果:在常氧條件下,6Gy的輻射劑量可顯著抑制MG-63細胞的增殖,給予MG-63細胞缺氧預處理后,輻射損傷對于細胞增殖的抑制作用被顯著減弱;6Gy的輻射劑量可顯著促進MG-63細胞的凋亡水平增加,給予MG-63細胞缺氧預處理后,輻射損傷對于細胞凋亡水平的促進作用被顯著減弱。結論:6Gy可有效抑制骨肉瘤MG-63細胞的增殖,增加細胞凋亡。缺氧微環(huán)境顯著升高骨肉瘤MG-63細胞增殖、凋亡率降低。缺氧微環(huán)境可能在增強骨肉瘤細胞放療抵抗中發(fā)揮重要作用。第二部分缺氧微環(huán)境誘導骨肉瘤細胞放療抵抗的機制目的:研究缺氧微環(huán)境誘導骨肉瘤細胞放療抵抗的具體機制,并對相關機制進行探討。方法:分別培養(yǎng)人骨肉瘤MG-63細胞和經過低氧條件預處理的人骨肉瘤MG-63細胞。采用重組腺病毒體外轉染,Western Blot方法,細胞免疫熒光技術檢測細胞DNA損傷(γ-H2AX),MitoSOX染色檢測線粒體ROS,檢測線粒體膜電位(MMP),流式細胞技術,MTT細胞增殖實驗,3-MA抑制自噬實驗、凋亡和自噬水平。結果:缺氧預處理可產生以下效應:1)誘導MG-63細胞中自噬水平的升高;2)通過降低細胞輻射損傷密切相關活性氧(ROS)水平達從而降低DNA損傷;3)降低線粒體ROS的產生進而保護了線粒體功能;4)利用自噬抑制劑抑制骨肉瘤細胞自噬后,缺氧微環(huán)境對骨肉瘤細胞耐受輻射損傷的保護作用消失。結論:缺氧微環(huán)境通過誘導骨肉瘤細胞發(fā)生自噬,提高了骨肉瘤細胞耐受輻射損傷的能力,因而導致骨肉瘤細胞發(fā)生放療抵抗。自噬可能介導了缺氧微環(huán)境對骨肉瘤細胞耐受輻射損傷的保護作用。第三部分缺氧誘導因子表達與骨肉瘤患者預后的相關性分析目的:研究缺氧誘導因子HIF-1、LC3-II及p62在人骨肉瘤組織中的表達,分析其與臨床病理的關系,并探討HIF-1在骨肉瘤發(fā)生和發(fā)展中的作用和意義。方法:采用免疫組織化學染色方法檢測89例(平均年齡為22歲)骨肉瘤組織中HIF-1的表達水平,并在只接受手術和放療的患者骨肉瘤標本中進一步檢測了LC3-II及p62的表達水平。Kaplan-Meier分析比較HIF-1、LC3-II及p62高表達、中表達和低表達患者生存率之間的差異。結果:HIF-1在骨肉瘤組織中低中高表達比例分別為20.2%(18/89)、24.7%(22/89)、55.1%(49/89),且HIF-1的表達與Enneking分期(P0.01)及遠處轉移(P0.01)顯著相關。而與其他臨床病理特征之間無明顯的關聯(lián)。LC3-Ⅱ在36例術后只接受放療的骨肉瘤患者中低中高表達比例分別為22.2%(8/36)、25%(9/36)、52.8%(19/36)。與此同時,52.8%(19/36)的骨肉瘤組織P62的表達水平較低,25%(9/36)中度表達,22.2%(8/36)高表達。且LC3-Ⅱ及P62的表達與Enneking分期(P0.01),遠處轉移(P0.01)及患者生存期顯著相關。而與其他臨床病理特征之間無明顯的關聯(lián)。結論:HIF-1、LC3-Ⅱ在骨肉瘤組織中高表達,而P62低表達。HIF-1、LC3-Ⅱ高表達,P62低表達提示臨床預后較差。骨肉瘤患者組織中自噬水平的高低與放療預后相關。
[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，

本文編號：2502758