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题名: 多参量细胞生理信息主动检测系统及应用研究
其他题名: Multi-parametric Cell Physiological Information Proactive Detection System and Application Research
作者: 张常麟
导师: 李广勇 ; 刘连庆
分类号: TB383
关键词: 平面膜片钳 ; 纳米操作机器人 ; 细胞生理信息 ; 机械门控离子通道 ; 离子通道电流
索取号: TB383/Z31/2014
页码: 116页
学位专业: 机械电子工程
学位类别: 博士
答辩日期: 2014-05-17
授予单位: 中国科学院沈阳自动化研究所
作者部门: 机器人学研究室
中文摘要: 细胞是一切生命体结构与功能的基本单元,一切的生命现象都与细胞的生长、发育、代谢、衰老、死亡等生物细胞信息密切相关,细胞信息获取在生物学研究上的地位越来越重要。目前,Biomarkers(物理、生化特性)已成为细胞生物学特性研究与表征的重要标记,细胞具有的各种生理信息对细胞生物学研究、新药研发、靶向医疗等具有重要意义。因此,如何在细胞水平上实现各种生理信息的获取手段已经成为多领域技术融合的研究热点。   细胞的Biomarkers具有多元性质,例如形态、硬度、酶化反应、特异性反应、离子交换等。其中细胞离子通道检测方法是获取细胞生理信息的重要手段。细胞所呈现的各种生理信息与环境变化和刺激密切相关,因而发展面向细胞的多元生理信息主动获取技术,对发现新的细胞Biomarkers,观测细胞对环境变化和药物施加的响应,研究分析细胞的活性变化、物质与信息交换机理等科学技术研究具有十分重要的意义。   针对细胞的多元生理信息主动获取技术的重大需求和已有技术的不足,本论文以膜片钳技术、机器人操控方法为基础,研究运用自动化、纳米操作、信息处理、系统集成技术,研究实现了多参量细胞生理信息主动检测系统构建;研究发展了以单细胞为对象的具有多个激励可控输入与多个响应检测量输出的细胞生理信息主动检测方法。并通过对活细胞的电压、机械门控离子通道和细胞膜杨氏模量检测实验,验证了系统的实用性和检测方法的可行性。本论文主要工作如下: (1) 平面膜片钳设计与实现技术研究   平面膜片钳主要用于全细胞离子通道实验条件建立与离子通道检测。通过研究分析平面膜片钳的机理,在自主设计基础上,结合飞秒激光加工技术与微电子机械系统(Micro-electromechanical Systems: MEMS)工艺,实现了具有细胞吸附、吉欧封接、破膜、电信号施加与提取功能的平面膜片钳系统,为开展电压与机械调控激励下的全细胞离子通道实验研究奠定了基础。   (2) 具有微力精准调控的生物型纳米操作机器人技术研究   基于纳米操作机器人系统技术,对生物型原子力显微镜(Atomic Force Mi-croscope: AFM)进行了具有实时视觉、触觉感知与反馈控制功能性构建研究,设计了针对细胞等生物样本的操作模块,使生物型原子力显微镜系统具有了可实现细胞刺激,力曲线收集及细胞保护参数设置等机器人化操作功能。   (3) 平面膜片钳与纳米操作机器人的系统集成研究   针对多参量细胞生理信息检测需求,开展了平面膜片钳、倒置荧光显微镜与纳米操作机器人相联合的实验系统集成研究。进行了相关功能软件设计、机构配合组装、电气连接、数据传输通讯等系统功能集成,解决了系统噪声消除、离子电流信号与外激励同步采集处理等关键问题,使该系统既具有精准的力、电激励操控和离子通道检测功能,又具有光学观测、荧光学信号识别与检测功能,可实现细胞生理信息检测的多参量输入与多种信息获取。   (4) 基于实验平台的离子通道实验研究   1) 开展了两类机械敏感性离子通道Piezos和TRPA1(Transient Receptor Po-tential A1)的多种外部机械力刺激离子通道实验,以及全细胞记录模式同步检测刺激产生的响应电流检测实验研究。实验结果表明,Neuro2A细胞上Piezos通道对AFM力刺激具有敏感性,而HEK293细胞上TRPA1通道对AFM力刺激不具有敏感性。   2) 以平面膜片钳探头电压作为刺激源,开展了对Neuro2A、HEK293、HaCaT等多种细胞的不同跨膜电压钳制与探针刺激力相结合的实验研究;对实验力曲线分析表明,Neuro2A细胞膜杨氏模量会随跨膜钳制电压的升高逐渐降低,而HEK293等其他细胞钳制电压的施加会改变其细胞膜的杨氏模量。 本文开展了集纳米操控技术、电生理学及细胞生物学为一体的多学科跨领域技术融合研究工作,研究成果为开展活细胞膜在机械与电可控激励下的生理特性信息提取与分析研究提供了有科学意义的系统支撑技术和实验方法,为开展机械门控离子通道的科学研究提供了一种有效技术手段,为细胞生物学、药物学等领域开展科学研究提供了有创新意义的技术途径。
英文摘要: Cell is the basic unit of all life structure and function. All life phenomena, in-cluding cell growth, cell development, metabolism, aging and death, have close rela-tionship with biological information of cell. Therefore, cell information acquisition is attracting more and more attention from biological researchers. Currently, biomarkers (physical, chemical and biological characteristics) have become key markers in cell biology study and cell characterization. A variety of physiological information of cells is of great significance to biology research, drug development, targeting medical, etc. Therefore, how to develop effective tools for acquiring a variety of physiological in-formation at the cellular level has become a hot multi-technology integrated topic. Cell biomarkers have diverse forms, such as shape, hardness, enzymatic reaction, specific reaction and ion exchange. Among them, ion exchange property reflects the physiological state of the cell. So, ion channel detection method is an important means to obtain cell physiological information. A variety of physiological information in cell is closely related to environmental changes and stimuli. Thus, it demands the development of diverse physiological information proactive acquisition technique for cell. Due to its great significance to science and technology renovations, such as the discovery of new cell biomarkers, observing cellular response to environmental changes and medication, analyzing cell activities and clarifying information exchange mechanism. This dissertation aims at meeting the desired needs of diverse physiological in-formation proactive acquisition techniques and eliminating the shortage of the already existed technologies. We achieved the construction of a multi-parametric cell physio-logical information proactive detection system with automation, nanomanipulation, information processing, and system integration technology based on the patch-clamp technique and the robot control method. The developed cell physiological information proactive detection approach has multiple controllable stimuli input and multiple de-tected responses output. Finally, in order to verify the practicality of the system and the feasibility of the detection method, experiments of detecting voltage-gated, mech-anosensitive ion channels and membrane Young's modulus of living cells are conducted. This dissertation’s main work is listed as following: (1) Launched a design and implementation research on planar patch clamp Planar patch clamp is mainly used for signal detection of whole-cell ion channel under controlled experimental physiological conditions. We developed the first set of planar patch clamp system through analyzing the work mechanism of planar patch clamp, which includes planar chip design and fabrication by MEMS technology and femtosecond laser processing, microfluidic chamber design and processing. The planar patch clamp system can achieve cell adsorbing, sealing, rupturing, electrical signal ap-plying and acquiring, which establishes the foundation for the whole-cell ion channels detection under the excitation of mechanical force and voltage regulation. (2) Bio-nanorobot with precise micro-force regulation technology research By employing the nanorobot technology, we updated the ordinary biological AFM with real-time visualization, tactile sense and feedback function. And we designed a unique biological operation module for biological sample including cells. With this module, robotic manipulating functions including cell stimulating, force curves collecting and parameters of cell protection setting are realized. (3) System integration research of planar patch clamp and nanorobot With the purpose of meeting the detection requirements of multi-parametric cell physiological information, we carry out system design and integration of planar patch clamp module and nanorobot module. To be specific, software design, hardware coop-eration, electrical signal connection and data transmission parts are integrated. Key problems, such as systematic noise removal and synchronous acquisition of ion current signal and external excitation, are solved. This system has not only the nanoscale mi-cro-force manipulation and ion channel current detection functions at special location, but signal recognition and detection of optic and fluorescence signals. All these features guarantees the realization of multi-parameter input and multiple responses acquisition of the cell physiological information detection system. (4) Experimental study of ion channel based the platform 1) We launch various ways of external mechanical force to two types of mecha-nosensitive ion channels (Piezos and TRPA1) based on experiment platform, a syn-chronously detect the response current activated by mechanical stimuli under whole-cell recording mode. The experimental results show that Piezos ion channels in Neuro2A cell are sensitive to AFM stimuli. While, TRPA1 channels in HEK293 cell are not sensitive to AFM stimuli. 2) Using the clamping voltage supplied by the planar patch clamp as a stimulus, experiments with varied transmembrane clamped voltage and different stimulating force are carried out on Neuro2A, HEK293and HaCat cells etc. Meanwhile, AFM tip probes force curves on the top of the cell membrane. By analyzing the acquired force curves, the changes of membrane’s mechanical properties can be reflected. The ex-perimental results show that the Young`s modulus of Neuro2A decreases gradually with the increase of the transmembrane clamping voltage. Whereas applying clamping voltage to HEK293 or other cells will change the related Young`s modulus. Combining nanomanipulation technology, electrophysiology and cell biology, this dissertation performed a multidisciplinary and interdisciplinary technique fusion re-search work. This research provides a scientific-significant supporting technology and experimental methods to carry out researches of physiological characteristics infor-mation extracting and analyzing of the living cell membrane under controllable me-chanical and electrical excitation. Our work provides an effective technical means for mechanosensitive ion channel scientific research and an innovative technical approach for cell biology and pharmacology research work.
语种: 中文
产权排序: 1
内容类型: 学位论文
URI标识: http://ir.sia.cn/handle/173321/14831
Appears in Collections:机器人学研究室_学位论文

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Recommended Citation:
张常麟.多参量细胞生理信息主动检测系统及应用研究.[博士学位论文].中国科学院沈阳自动化研究所.2014
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