【作者】 陈丽霞；

【导师】 齐燕飞；

【作者基本信息】 吉林大学 ， 劳动卫生与环境卫生学， 2023， 硕士

【摘要】 癌症是全球性重大公共卫生问题,严重威胁人类健康。高能电离辐射与生物分子相互作用直接杀死肿瘤细胞,也能通过肿瘤微环境中水分子的辐射分解产生自由基间接杀死肿瘤细胞。但由于肿瘤的特殊微环境,如肿瘤乏氧,导致肿瘤细胞对放射线的抗性增强,放射治疗效果不明显。因此,筛选高效、低毒的放射增敏剂对于提高肿瘤的放射敏感性具有重要意义。多金属氧酸盐（简称多酸）是由高原子序数金属元素特别是钼,钒,钨元素通过氧连接而成的多阴离子簇,是一类结构确定的纳米尺度的无机簇合物,粒径一般在0.5-6nm之间。已有研究表明,含有高原子序数金属具有很强的X射线沉积能力。因此,多酸有望作为一种新型肿瘤放射增敏剂。研究目的:合成多酸及多酸复合物,对其组成、结构和形貌,生理稳定性进行表征,以人结肠癌细胞系（Human Colorectal Carcinoma Cells,HT-29）为模型,明确多酸及多酸复合物联合X射线对其放射增敏作用,阐明多酸及多酸复合物联合X射线放射敏感性的机制,为新型放射增敏剂提供科学依据。研究方法:1.制备合成多酸化合物Na₆V₁₀O₂₈和K₃Na₄H₂(Gd W₁₀O₃₆)·2H₂O。以K₃Na₄H₂(Gd W₁₀O₃₆)·2H₂O为母体,通过优化多巴胺与钨钆酸盐的质量比和反应溶剂体积,水热法制备聚多巴胺包覆的钨钆酸盐纳米复合物(Gd W₁₀@PDA),利用聚多巴胺表面官能团在Gd W₁₀@PDA表面共价偶联过氧化氢酶（CAT）,制备钨钆酸盐过氧化氢酶复合物(Gd W₁₀@PDA-CAT)。2.采用透射电子显微镜（TEM）、紫外分光光度计（Uv-Vis）、傅立叶红外光谱仪（IR）、马尔文粒度分析仪,热重分析等对多酸和多酸复合物的形貌,组成进行表征。3.利用紫外吸收法研究Gd W₁₀@PDA-CAT过氧化氢酶的活性,硫氰酸钾-三氯化钛分光光度法评价Gd W₁₀@PDA-CAT稳定性。4.选择人结肠癌细胞系HT-29作为细胞模型,利用噻唑蓝比色法（MTT）研究X射线和多酸及多酸复合物对肿瘤细胞增殖的影响。通过免疫荧光法检测X射线和多酸及多酸复合物对HT-29细胞存活及形态学的影响。利用流式细胞仪研究X射线和多酸及多酸复合物对肿瘤细胞凋亡的影响。利用克隆集落实验研究多酸及多酸复合物联合X射线对肿瘤细胞增殖的抑制作用,计算放射增敏比。通过免疫荧光法测定多酸及多酸复合物联合X射线对HT-29细胞中γ-H₂AX和活性氧ROS的水平。研究结果:1.十钒酸盐Na₆V₁₀O₂₈和钨钆酸盐复合物Gd W₁₀@PDA-CAT结构、组成明确,在生理条件下具有良好的稳定性。2.MTT实验结果表明,十钒酸盐Na₆V₁₀O₂₈和钨钆酸盐复合物Gd W₁₀@PDA-CAT对人结肠癌细胞HT-29增殖的抑制作用具有浓度和剂量依赖效应。Na₆V₁₀O₂₈作用24小时半数有效抑制浓度(IC₅₀)为8.994μg/m L。钨钆酸盐复合物对HT-29细胞的抑制作用较低,24小时半数有效抑制浓度为81.03μg/m L。在50μg/m L浓度剂量下,Gd W₁₀@PDA-CAT联合放射对HT-29细胞有明显放射增敏作用。Hoechst33342/PI染色表明,多酸联合X射线处理的HT-29细胞发生大量的凋亡和坏死。3.流式细胞仪分析结果表明,将HT-29细胞与两种多酸孵育后给予X射线辐照,联合处理组细胞具有更多的凋亡及坏死,差异有统计学意义。4.克隆集落形成实验结果表明,两种多酸都具有明显的放射增敏效果,与X射线联合处理能显著抑制HT-29细胞集落形成。钨钆酸盐复合物的放射增敏比为3.7,十钒酸盐的放射增敏比为8.52。5.免疫荧光法结果表明,多酸与X射线联合处理组HT-29细胞的DNA双链断裂标志物γ-H₂AX的表达水平显著高于其他对照组,DNA双链发生了明显的断裂,HT-29细胞凋亡。6.活性氧探针检测活性氧生成情况结果表明,HT-29肿瘤细胞与多酸孵育并联合X射线照射后表现出较强的荧光信号,表明有大量的活性氧生成,从而诱导肿瘤细胞的死亡。研究结论:1.成功合成了多酸和多酸复合物,两种物质结构明确,性质稳定。2.多酸和多酸复合物能够降低HT-29细胞的存活率,具有一定的剂量-反应关系。3.含有Gd、W和V高Z元素的多酸和多酸复合物,能够在肿瘤细胞中沉积大量的辐射剂量,增强X射线与DNA的相互作用,导致大量的DNA双链断裂,增加活性氧水平,从而增强肿瘤细胞放射敏感性。更多还原

【Abstract】 Cancer is a major global public health problem and a serious threat to human health.High energy ionizing radiation can kill tumor cells directly by interacting with biomolecules and indirectly by generating free radicals from radiolysis of water molecules in tumor microenvironment.However,due to the special microenvironment of tumor,such as tumor hypoxia,the resistance of tumor cells to radiation is enhanced,and the effect of radiotherapy is not obvious.Therefore,it is of great significance to select highly effective and low-toxicity radiosensitizers to improve the radiosensitivity of tumors.Polyoxometalate（POMs for short）are polyanion clusters formed by high ordinal number metal elements,especially molybdenum,vanadium,and tungsten,connected by oxygen.They are a kind of inorganic clusters with nanoscale structure and particle size generally between 0.5-6nm.It has been shown that metals containing high ordinal number have strong X-ray deposition ability.Therefore,POM is expected to be used as a new sensitizer for radiation therapy of tumor.Objective:POM and POM complex were synthesized,and their composition,structure,morphology,and physiological stability were characterized.Using human colon cancer cell line HT-29 as the model,the radiosensitization effects of POM and POM complex combined with X-ray on POM and POM complex combined with X-ray were clarified,and the mechanism of radiosensitization of POM and POM complex combined with X-ray was elucidated,to provide scientific basis for new radiosensitizer.Methods:1.The POMs Na₆V₁₀O₂₈ and K₃Na₄H₂(Gd W₁₀O₃₆)·2H₂O are synthesized.Using K₃Na₄H₂(Gd W₁₀O₃₆)·2H₂O as the matrix,the dopamine-tungstate gadolinium nanocomplex(Gd W₁₀@PDA)was prepared by hydrothermal method by optimizing the mass ratio of dopamine to tungstate gadolinium and the volume of reaction solvent.A tungstate gadolinium catalase complex(Gd W₁₀@PDA-CAT)was prepared by covalently coupling catalase（CAT）to the surface of Gd W₁₀@PDA using a polydopamine surface functional group.2.Transmission electron microscope（TEM）,ultraviolet spectrophotometer（UV-Vis）,Fourier infrared spectrometer（IR）,Malvern particle size analyzer,thermogravimetric analysis were used to characterize the morphology and composition of POM and POM complex.3.The catalase activity of Gd W₁₀@PDA-CAT was studied by ultraviolet absorption,and the stability of Gd W₁₀@PDA-CAT was evaluated by potassium thiocyanate-titanium trichloride spectrophotometry.4.Human colon cancer cell HT-29 was selected as the cell model to study the effects of X-ray and POM and POM complex on tumor cell proliferation by thiazolam colorimetry（MTT）.The effects of X-ray and POM and POM complex on survival and morphology of HT-29 cells were determined by immunofluorescence method.The effects of X-ray,POM and POM complex on apoptosis of tumor cells were studied by flow cytometry.The inhibitory effect of POM and POM complex combined with X-ray on tumor cell proliferation was studied by clonal colony experiment,and the radiosensitization ratio was calculated.The levels ofγ-H₂AX and ROS in HT-29 cells affected by POM and POM complex combined with X-ray were determined by immunofluorescence method.Results:1.Synthesis of decadadate Na₆V₁₀O₂₈ and tungstate gadolinium complex Gd W₁₀@PDA-CAT,the two substances have clear structure and composition,and have good stability under physiological conditions.2.Decavadate Na₆V₁₀O₂₈ and tungstate gadolinium complex Gd W₁₀@PDA-CAT showed concentration-and dose-dependent inhibition of HT-29 proliferation in human colon cancer cells.The median effective inhibitory concentration(IC₅₀)of Na₆V₁₀O₂₈for 24 hours was 8.994μg/m L,the inhibitory effect of tungstate gadolinium complex on HT-29 cells was low,and the median inhibitory concentration was 81.03μg/m L at 24hours.Gd W₁₀@PDA-CAT had no inhibitory effect on HT-29 cells at the concentration of 50μg/m L,but showed radiosensitization.Hoechst33342/PI staining showed that the tumor cells treated with two POMs combined with X-ray showed strong red fluorescence signal,and the cells showed many apoptosis and necrosis.Hoechst33342/PI staining showed that tumor cells treated with two kinds of POMs combined with X-ray showed strong red fluorescence signal,and many apoptosis and necrosis occurred.Hoechst33342/PI staining showed that tumor cells treated with two kinds of POMs combined with X-ray showed strong red fluorescence signal,and many apoptosis and necrosis occurred in the cells.3.The results of flow cytometry analysis showed that HT-29 cells were incubated with two POMs and then subjected to X-ray irradiation,and the cells in the group were treated with more apoptosis and necrosis,and the difference was statistically significant.4.The results of colony formation experiment showed that both POMs had obvious radiosensitization effect,and the combination treatment with X-ray could significantly inhibit the formation of cell colonies.The radiosensitization ratio of gadolinium ungstate complex was 3.7,and that of decadadate was 8.52.5.The expression level of DNA double-strand break markerγ-H₂AX in tumor cells after different treatments was detected by immunofluorescence method.The results showed that the content of POMs and X-ray combined treatment group was higher than that of other single treatment groups,indicating that the DNA double-strand break occurred significantly,leading to tumor cell apoptosis.6.Reactive oxygen species（ROS）generation in tumor cells in different treatment groups was detected by ROS probes.Tumor cells incubated with POMs and irradiated by X-rays showed strong fluorescence signals,indicating a large amount of ROS generation,thus inducing tumor cell death.Conclusion:1.Successfully synthesized POM and POM complex,the two substances have clear structure and stable properties.2.POM and POM complex can reduce the survival rate of HT-29 cells and have a certain dose-response relationship.3.POM and POM complex containing Gd,W and V high-Z elements can deposit large amounts of radiation in tumor cells,enhance the interaction between X-ray and DNA,lead to a large number of DNA double-strand breaks,increase the level of ROS,and thus enhance the radiosensitivity of tumor cells.更多还原