【作者】 王英；

【导师】 赵祥伟；

【作者基本信息】 东南大学 ， 生物医学工程， 2022， 硕士

【摘要】 空间转录组学是继单细胞转录组学后的又一研究热潮,它旨在获取基因表达信息的同时保留样本的原始空间位置,成为研究组织空间异质性的强有力手段。其中,基于显微切割的空间转录组技术中应用最为广泛的是激光捕获显微切割(Laser capture microdissection,LCM)技术,它利用激光精准切割组织切片上的感兴趣区域(Region of Interest,ROI),并与福尔马林固定石蜡包埋(Formalin fixed paraffin embedded,FFPE)组织兼容。但由于LCM设备价格昂贵、操作相对复杂、样本收集率有限、可能损伤核酸等缺陷,急需一种替代方法来采集特定位点的微区样本,满足低通量空间转录组学研究的采样需求。在显微切割技术充分调研和预实验结果的基础上,本文设计并制作了一种基于玻璃微针的组织切片微区样本采集装置,在使用过程中不断优化采样方案,并探究了装置的适用条件和性能。论文的主要内容如下:1.开发了一种基于玻璃微针的组织微区样本采集装置。选用玻璃微针作为显微切割工具,其原理是用尖锐细小的针尖与切片接触时产生的压力来切割捕获组织的圆形区域。采集装置主要包括了电动载物台、微针及控制模块、倒置荧光显微成像模块和侧方显微观察模块,能够实现组织切片上ROI样本的选择、显微切割和转移。2.优化了组织微区样本的采集方案。转录组学实验处理的是易降解的信使RNA(m RNA),微针采样方案的优化可以提高转录本质量。在此,确定了制备特定直径微针的程序参数以提高微针的利用率;采用了聚二甲基硅氧烷(Polydimethylsiloxane,PDMS)薄膜和等离子键合来制备缓冲玻片,防止微针断裂的同时提高样本采集成功率;确定了通过微针采样前吸取少量裂解液随后立即排出的方式来转移样本,减少核酸损失。3.实现了冰冻切片的高效采样,并探究了装置的适用条件和性能。对冰冻组织切片进行采样和测序分析,验证了装置的可靠性。此外,实验结果还表明了该装置适用于冰冻组织切片,空间分辨率达到单细胞尺度(10μm),单个样本采集时间为2 min,采样成功率高达91.7%。自主设计的基于玻璃微针的组织切片微区样本的采集装置,在实验方案的优化后性能得到提升,可在明场和荧光观察下收集冰冻组织微区样本,具有单细胞尺度分辨率、时间效率高、样本收集成功率高、制作成本较低等优势,有望成为LCM系统的替代装置,为研究组织样本的时空异质性提供了又一高效方法。更多还原

【Abstract】 Spatially resolved transcriptomics has become another research hotspot after single-cell transcriptomics,which obtains gene expression information as well as the original spatial location of sample.Spatial transcriptome technologies have become powerful means to study the spatial heterogeneity of biological tissues.Among current spatial transcriptome technologies based on microdissection,the most widely used is LCM,which utilizes laser to precisely cut ROI of tissue section and is compatible with FFPE tissue.In view of the present shortage of LCM system,such as the expensive price,relatively complicated procedure,limited sample collection rate and possible nucleic acid damage,an alternative method is urgently needed to acquire micro-region samples at specific sites on tissue section for meeting the sampling needs of low-throughput spatial transcriptomics research.On the basis of full investigation of microdissection technology and pre-experimental results,this paper designs and produces a glass microneedle-based device for acquiring microregion sample on tissue section.Subsequently,the sampling protocol is optimized and the applicable conditions and performance of the device are explored during use.The main contents in this paper are as follows:1.A microneedle-based acquisition device of tissue micro-region sample is developed.Glass microneedles are used as the microdissection tool.Once the tip of sharp needle contacts with the tissue section,the micro-region of tissue section in the circular area is cut off under pressure.The designed device mainly includes a motorized stage,a microneedle and its control module,an inverted fluorescence microscopic imaging module and a lateral microscopic observation module,which can realize the selection of ROI sample on tissue sections,microdissection and transfer.2.Tissue micro-sample acquisition scheme is optimized.Transcriptomics experiments deal with easily degradable m RNA,and the optimization of sampling protocol can better transcript quality.Here,the preparation parameters of microneedles with specific diameter are obtained,which increase the utilization rate of microneedles;the buffer slides made by PDMS film and plasma bonding is selected to prevent microneedles from breaking and improve sampling success rate;the means of sample transfer which is that microneedles suck a small amount of lysis before sampling and drain micro-sample immediately is determined to reduce the loss of nucleic acid.3.Highly efficient sampling of frozen sections was achieved,and the applicable conditions and performance of the device were explored.Sampling and sequencing analysis of frozen tissue sections verify the reliability of the device.Furthermore,the results show that the device is suitable for frozen tissue sections,its spatial resolution reaches the single-cell scale(10 μm),its collection time of a sample is 2 min,and its success rate of sample capture is as high as 91.7%.The self-designed glass microneedle-based acquisition device for micro-region tissue has improved performance in the continuous optimization of operation process.It can be applied to frozen tissue micro-samples acquisition under brightfield and fluorescence observation.In addition,it owns the advantages with single-cell scale resolution,high time efficiency,high success rate and low cost,which make it possible to become a potential device to the LCM system.The device becomes another efficient method for studying the spatiotemporal heterogeneity of tissue samples.更多还原