【作者】 张威；

【导师】 王平；

【作者基本信息】 浙江大学 ， 生物医学工程， 2011， 博士

【摘要】 味觉是人体重要的生理感觉,五种基本味道分别为酸、咸、苦、甜和鲜。溶解于唾液中的味觉物质被口腔中的味觉细胞捕获,产生特异性味觉响应。味觉响应信息经过外周系统和中枢系统的时空整合和处理,最终产生味觉。分子生物学和脑成像等现代科学技术的进步大大推动了味觉研究的发展。以往味觉电信号的获取一直依赖于有创的方式,直接对味觉细胞或神经纤维进行记录,而且同步记录的位点非常有限。本研究基于光寻址电位传感器(light addressable potentiometric sensor, LAPS)和微电极阵列传感器(microelectrode array, MEA)构建了味觉细胞传感器,采用无损的胞外记录方式,实现对味觉受体细胞和味觉神经网络的实时检测。着重分析了酸敏感细胞对pH变化的机理,建立了全细胞胞外模型。在MEA上成功培养了味觉神经网络,并利用MEA多通道同步记录,进一步研究了味觉信息在外周及中枢味觉系统的时空传导过程。本论文的主要内容和贡献如下：1、提出了味觉细胞传感器相容性设计方法。针对味觉单细胞和神经网络在传感器上的固定提出新方案。利用自组装单分子层(self-assembled monolayer, SAM)技术修饰MEA表面,可以有效的引导细胞生长在目标测试部位。微接触印章(microcontact printing, MCP)技术可用于在传感器表面固定神经网络,人工诱导细胞网络图形化生长。使用循环伏安法、交流阻抗测试以及荧光标记等手段对修饰后的表面进行评估,表明修饰后的传感器表面具有良好的生物相容性,能维持细胞生理活性,可定向诱导细胞生长。2、深入研究了味觉受体细胞在传感器上的培养和固定。在LAPS仿生味觉芯片的基础上,记录了味觉初级感受细胞对混合味质刺激的响应信号,表明了味觉受体细胞对溶于水中的酸、甜、苦、咸和鲜等味质具有特异性响应,发现了在7-10 Hz存在味觉特征响应频率。3、进一步发展和完善了味觉细胞模型。在酸敏感通道ASIC和PKDL独立模型的基础上建立酸敏感细胞的全细胞模型,并利用全细胞模型仿真细胞在不同pH的盐酸刺激下的响应。成功将Ⅲ型味觉细胞培养于MEA芯片表面,并使用不同的酸刺激,记录到了细胞对不同浓度酸溶液的响应,并与全细胞模型仿真结果进行比较,对模型进行说明。4、实现了味觉神经网络传感器的设计和味觉信息的时空分析。在MEA器件表面成功固定了味觉神经组织,记录并分析其在神经递质5-HT刺激下的响应。利用MCP技术进一步人工诱导味觉中枢神经细胞在离体条件重建神经细胞网络。使用多通道MEA同步记录备节点神经元细胞对神经递质5-HT的响应,进一步分析了味觉信息在味觉中枢神经系统的时空传导和信号处理过程。本论文利用SAM和MCP技术在传感器上有效地引导味觉细胞和味觉神经细胞网络的生长,为味觉外周系统和中枢系统信息传导的研究提供了有效的手段。更多还原

【Abstract】 Taste sensation is one of the basic physical feelings of human. There are five basic taste qualities, including sourness, saltiness, bitterness, sweetness and umami. Taste substance dissolved in saliva is captured by taste cells in oral cavity, which generate specific response uniquely. Taste information is firstly integrated in periphery system and then processed in central system in spatio-temporal manner to a final taste recognization.Taste research is dramatically promoted due to the development of modern scientific technologies, such as molecular biology, brain imaging, and so on. In the past, electric signals of taste cell were obtained by some invasive ways, in which cells or neural fibers were directly clamped. There were also few sites for synchronous data recording. In this thesis, taste cell-based sensors were constructed on the basis of light addressable potentiometric sensor (LAPS) and microelectrode array (MEA). It was able to perform a real-time extracellular measurement on taste receptor cells and taste neuronal network in a noninvasive way. Response mechanism of sour perception cells was profoundly analyzed under different pH values. A whole-cell extracellular model for sour perception cells was successfully built. Taste neuronal network was reconstructed on MEA in an artificial way, and multi-site neurons on the node were recorded synchronously. Further studies on spatio-temporal transduction process of taste information in periphery and central systems were carried on.The main contents and contributions of this thesis are as follows:1. Compatibility design methods for taste cell-based sensors were presented. Novel methods for single cell and neuronal network immobilizations on sensors were proposed. Self-assembled monolayer (SAM) technology was applied on MEA surface treatment, and cells were attracted to land on the target regions for test efficiently. Microcontact printing (MCP) technology was introduced to culture neuronal network on the surface of sensors, which was induced spreading according to artificial patterns. Cyclic voltammetry (CV), AC impedance test and fluorescence labeling were utilized to evaluate surface characteristics after treatment. Results indicated the surface exhibited well biocompatibility, which could induce cells immobilization in orientation as well as keeping good physiological activities. 2. Taste receptor cells immobilization on chips was profoundly studied. Responses of taste receptor cells under taste substances mixture stimulus were measured on the basis of LAPS biomimetic taste chip. Primary taste cells responded to sour, sweet, bitter, salty and umami qualities in solution specifically. It was discovered that it existed 7～10 Hz specific frequency component in frequency domain.3. A model for taste receptor cells was further developed. Extracellular model of sour perception cells was constructed. A whole-cell model was presented based on the respective models of sour perception ASIC and PKDL channels. Simulations of the responses under different pH values of hydrochloric acid solution were carried out. TypeⅢtaste cells were cultured on MEA surface successfully. The response under different sour concentrations were recorded and compared with simulation results calculated by the whole-cell model.4. Design of taste cell network-based sensor and spatio-temporal analysis of taste information were achieved for the first time. Taste neural tissue was managed to immobilize on MEA surface and the responses to serotonin (5-HT) were recorded and analyzed. Furthermore, MCP technology was utilized to induce taste central neuronal network reconstruction artificially in-vitro. The responses of neurons on the node to neurotransmitter (5-HT) were acquired via MEA multi-site recording synchronously. Spatio-temporal transduction of taste information in taste central system and signal processing were further analyzed.In the thesis, SAM and MCP technologies were employed inducing taste cells and taste neuronal network immobilization on the surface of biosensor. It presented an efficient method for the research on information transduction in taste periphery and central systems.更多还原