【作者】 李萍；

【导师】 韩琳；

【作者基本信息】 山东大学 ， 电子科学与技术， 2023， 硕士

【摘要】 近年来,ZnO纳米棒已被广泛研究用于癌细胞的特异性杀伤,其杀伤机制已被初步证明:锌离子的溶解和过量活性氧物质的产生使细胞产生氧化应激反应,进而促使癌细胞凋亡。ZnO纳米棒在癌细胞中的主动靶向能够避免对健康细胞造成损害,减少非特异性杀伤,有望成为新型抗癌材料。然而,针对其抗癌效果的研究只局限于检测凋亡基因及相应蛋白,忽视了细胞作为一个复杂生物体,各项生理活动和功能是彼此相关、相互作用的。此外,已有的研究均聚焦于群体细胞分析,平均化了细胞功能,忽视了细胞异质性在肿瘤进化中的重要作用,遗漏了癌症治疗的部分关键信息。本文针对这一课题,利用基于微电子技术的微流控芯片和生物传感纳米材料氧化石墨烯量子点(GOQDs),以传递功能信息的细胞因子作为分析对象,兼顾群体水平和单细胞水平研究了 ZnO纳米棒对乳腺癌细胞的抑制作用。本文的主要研究内容如下:(1)通过微流控生物芯片研究了 ZnO纳米棒对乳腺癌群体细胞的抑制作用。ZnO纳米棒采用水热法制备并分别用于乳腺癌的典型细胞系MDA-MB-231和MCF-7细胞培养。材料表征实验证明该棒体是垂直生长的单晶六方纳米棒,棒体直径为152.6±28.2 nm,高度约为1240nm。基于光刻技术制备了微流控芯片,利用纳米材料自组装技术制备了 GOQDs传感检测基底,二者共同实现了对群体细胞12种细胞因子的高通量检测。ZnO纳米棒促进了 MDA-MB-231和MCF-7细胞的HSP70因子分泌,抑制了其他11种细胞因子的分泌,反映了纳米棒对癌细胞的生长和转移具有抑制作用。(2)利用单细胞芯片研究了 ZnO纳米棒对乳腺癌细胞异质性和细胞亚群功能的影响。基于光刻技术制备了单细胞芯片,用于MDA-MB-231和MCF-7单细胞培养;捕获抗体条形码基底用于单细胞因子的捕获和收集。通过t-SNE方法和基于社区的聚类方法对所有单细胞数据进行降维和分类,将每个单细胞对12种细胞因子的分泌量可视化,以形成不同表型和功能的多个细胞亚群。Spearman相关性分析证明ZnO纳米棒改变了IFN-γ、TNF-α和Granzyme B因子的功能及亚群间相关性,揭示了纳米棒对转移型功能亚群的低抑制性、对增殖型功能亚群的高抑制性,暗示了 ZnO纳米棒对强浸润性乳腺癌细胞系MDA-MB-231的杀伤和治疗存在一定的风险。本论文首次在单细胞分辨率下对ZnO纳米棒的抑癌作用和效果进行了研究和探讨,补充了群体水平层次的研究结果,为ZnO纳米棒在癌症治疗上的应用提供了合理的风险分析,对纳米材料在癌症治疗、病理分析等生物医学上的应用研究有重要的借鉴意义。更多还原

【Abstract】 Zinc oxide nanorods(ZnO NRs)have garnered significant attention in recent years due to their ability to selectively target and kill cancer cells.The mechanism behind this process has been preliminarily proven:the dissolution of zinc ions and excess generation of reactive oxygen species induces oxidative stress,which ultimately leads to cancer cell apoptosis.The active targeting of ZnO NRs to cancer cells minimizes damage to healthy cells and reduces nonspecific killing,making them a promising new material for anticancer treatment.However,previous studies have focused on the analysis of apoptotic genes and corresponding proteins in cells,overlooking the fact that cells are complex organisms with interconnected physiological activities and functions.Additionally,prior researches have primarily utilized population-cell analysis,which failed to capture the critical role of cell heterogeneity in tumor evolution and overlooked essential information regarding cancer treatment.Herein,this.thesis utilized microfluidic chips based on microelectronic technology and bio-sensing nanomaterials,graphene oxide quantum dots(GOQDs),to analyze functional cytokines secrected from cells.It investigates the inhibitory effect of ZnO nanorods on breast cancer cells at both the population and single-cell levels.The main research contents of this thesis are as follows:The inhibitory effect of ZnO NRs on breast cancer population cells was studied by microfluidic biochips.MDA-MB-231 and MCF-7 cells were severally cultured with hydrothermally prepared ZnO NRs,which were single-crystal hexagonal nanorods with a diameter of 152.6 ± 28.2 nm and a vertical height of approximately 1240 nm,as confirmed by multiple material characterization experiments.The microfluidic chips were fabricated using photolithography technology,and GOQDs sensing detection substrate were prepared using nanomaterial self-assembly technology,which were used to detect 12 cytokines in a highthroughput manner.ZnO NRs promoted the secretion of HSP70 factors from MDA-MB-231 and MCF-7 cells while inhibiting the secretion of other 11 cytokines,indicating that cells were prevented from proliferating and metastasizing.The effect of ZnO NRs on breast cancer cell heterogeneity and cluster functions was investigated through single-cell microchips.The microchips used for MDA-MB-231 and MCF7 single cells culture were prepared using photolithography technology.The capture antibody barcode substrates were utilized to collect cytokines secreted by individual cells.The t-SNE and community-based clustering methods were employed to analyze the resulting data and visualize the secretion of 12 cytokines,thus forming multiple cell clusters with different plienotypes and functions.Spearman analysis indicated that ZnO NRs altered the functions of IFN-γ.TNF-α.and Granzyme B.and weakened the correlation between elusters,suggesting a greater inhibitory effect on proliferative functional clusters than metastatic functional clusters.These findings implied that ZnO NRs could pose a risk to the treatment of highly invasive breast cancer cells,such as MDA-MB-231.This thesis provided,for the first time,a single-ceIl resolution analysis of the tumor suppressor effect of ZnO NRs,supplementing previous researches conducted at the populationcell level.The findings offered a reasonable risk analysis for the potential application of ZnO NRs in cancer therapy.This thesis holds significant reference value for the application of nanomaterials in biomedicine,particularly in the fields of cancer treatment and pathological analysis.更多还原