【作者】 张艳；

【导师】 林少鹏； 付琪镔；

【作者基本信息】 中山大学 ， 核能科学与工程， 2022， 硕士

【摘要】 到目前为止,肿瘤依然是造成人类死亡、危害人类健康的重大疾病之一。放射治疗是目前癌症治疗的主要手段之一。DNA是电离辐射作用于生物目标的主要靶向分子。电离辐射诱导的DNA损伤是研究放射生物机制的核心,也是癌症治疗起效的关键机制。因此,电离辐射的DNA损伤响应过程一直是国内外学者研究的焦点。近年来的研究发现,细胞力学特性会影响很多重要的细胞功能与生理过程,例如细胞的运动、侵袭和转移以及基因组的完整性。然而,辐射诱导的DNA损伤响应是如何影响细胞力学性质以及细胞力学性质又如何影响DNA损伤尚不明确。一个重要的原因是缺乏合适的测量细胞力学性质的工具。加之,电离辐射会增强细胞的粘附性并导致细胞扁平化,这会影响在贴壁状态下测量细胞力学性质的准确性。为此,本文自主设计和建立了基于超声驻波技术的细胞力学测量装置,可以实现在悬浮状态下快速、无损地测量癌细胞地可压缩性(compressibility)。可压缩性是细胞力学性质的重要参数,是细胞整体模量的反映。结合免疫荧光法、细胞活性实验等生物学方法,本文系统地研究了 X射线对癌细胞力学性质的影响,主要工作内容和结论如下:1.建立超声驻波微流芯片装置。装置能够对流体中细胞进行无接触式操纵进而测量细胞可压缩性。测量细胞可压缩性过程中对细胞损伤小,且测量速度快,通量高。2.基于超声驻波微流装置测量多种癌细胞系的可压缩性。侵袭性强的乳腺癌细胞系MDA-MB231较非侵袭性的MCF-7可压缩性大(即更软),实现对不同侵袭能力的癌细胞可压缩性的测量。乳腺癌细胞系MDA-MB231、肺腺癌细胞系A549与结肠癌细胞系HCT116在可压缩性和尺寸方面存在差异,细胞系按可压缩性由大到小排列为 MDA-MB231>A549>HCT116。3.X射线影响细胞力学性质。经过X射线辐照后3小时,细胞的可压缩性显著增大,并且可压缩性的变化与辐射剂量以及细胞类型有关。4.辐射诱导的DNA损伤响应对细胞力学性质影响及机制。抑制DNA损伤响应过程抑制了辐射诱导的可压缩性的变化。进一步对异染色质结构以及细胞骨架蛋白进行了研究。辐射后3h异染色质的荧光强度下降,细胞核中染色质结构发生去凝聚,但细胞骨架蛋白在荧光强度和组织分布方面没有发生显著的变化。由此揭示了辐射诱导的可压缩性变化与DNA损伤响应导致的细胞核中染色质发生去凝聚有关。5.细胞力学性质改变影响辐射诱导DNA损伤响应。通过在X射线辐照前加入抑制肌球蛋白的药物blebbistatin,细胞核受到的应力减弱,细胞可压缩性增大,而且辐射诱导的53BP1 foci和γH2AX foci显著减少,揭示了细胞可压缩性的增加会减弱辐射诱导的DNA损伤。通过本论文的研究,实现了对不同种类、不同侵袭能力的癌细胞的可压缩性测量。并且揭示了 X射线诱导的DNA损伤响应与癌细胞力学性质之间的相互调控机制,为深入理解电离辐射诱导的生物效应提供新的思路。更多还原

【Abstract】 Up to now,tumor is still one of the major diseases that cause human death and endanger human health.Radiation therapy is one of the main methods of cancer treatment at present.DNA is the main target molecule for ionizing radiation to act on biological targets.Ionizing radiation-induced DNA damage is central to the study of biological mechanisms of radiation and a key mechanism for the efficacy of cancer therapy.Therefore,the DNA damage response process of ionizing radiation has always been the focus of research by scholars at home and abroad.Recent studies have found that cell mechanical properties affect many important cellular functions and physiological processes,such as cell motility,invasion and metastasis,and genome integrity.However,how the radiation-induced DNA damage response affects cellular mechanical properties and how cellular mechanical properties affect DNA damage remains unclear.An important reason is the lack of suitable tools for measuring the mechanical properties of cells.In addition,ionizing radiation enhances cell adhesion and causes cell flattening,which affects the accuracy of measuring cell mechanical properties in the adherent state.To this end,this paper independently designed and established a cell mechanics measurement device based on ultrasonic standing wave technology,which can quickly and non-destructively measure the compressibility of cancer cells in a suspended state.Compressibility is an important parameter of the mechanical properties of cells and is a reflection of the overall modulus of the cell.Combined with biological methods such as immunofluorescence and cell viability experiments,this paper systematically studies the effects of X-rays on the mechanical properties of cancer cells.The main work and conclusions are as follows:1.Establish an ultrasonic standing wave microfluidic chip device.The device enables non-contact manipulation of cells in fluids to measure cell compressibility.In the process of measuring cell compressibility,the damage to cells is small,and the measurement speed is fast and the throughput is high.2.Measure the compressibility of various cancer cell lines based on an ultrasonic standing wave microfluidic device.The aggressive breast cancer cell line MDA-MB231 is more compressible(ie,softer)than the non-invasive MCF-7,enabling measurement of the compressibility of cancer cells with different invasive abilities.Breast cancer cell line MDA-MB231,lung adenocarcinoma cell line A549 and colon cancer cell line HCT116 have differences in compressibility and size.3.X-rays affect the mechanical properties of cells.After 3 hours of X-ray irradiation,the compressibility of cells increased significantly,and the change of compressibility was related to radiation dose and cell type.4.The effect and mechanism of radiation-induced DNA damage response on cellular mechanical properties.Inhibition of DNA damage response processes suppressed radiation-induced changes in compressibility.Heterochromatin structure and cytoskeletal proteins were further studied.The fluorescence intensity of heterochromatin decreased 3 h after irradiation,and the chromatin structure in the nucleus decondensed,but the fluorescence intensity and tissue distribution of cytoskeletal proteins did not change significantly.This revealed that radiation-induced changes in compressibility were associated with decondensation of chromatin in the nucleus in response to DNA damage.5.Changes in cellular mechanical properties affect the response to radiation-induced DNA damage.By adding the myosin-inhibiting drug blebbistatin before X-ray irradiation,the stress on the nucleus was reduced,the cell compressibility was increased,and the radiation-induced 53BP1 foci and yH2AX foci were significantly reduced,revealing that the increase in cell compressibility would Attenuates radiation-induced DNA damage.Through the research in this paper,the compressibility measurement of cancer cells with different types and different invasive abilities is achieved.Moreover,the inter-regulation mechanism between X-ray-induced DNA damage response and the mechanical properties of cancer cells was revealed,providing new ideas for in-depth understanding of ionizing radiation-induced biological effects.更多还原