味觉细胞模型及仿生味觉细胞网络传感器的研究

发布时间：2018-06-04 14:01

  本文选题：味觉细胞电生理模型 + PKD1L3/2L1通道　； 参考：《浙江大学》2010年博士论文

【摘要】： 味觉感受细胞是味觉敏感的基本单元,实现味觉中化学-电信号转换等重要的前期信息处理功能。研究表明酸、甜、苦、咸和鲜味味质能刺激味觉感受细胞产生动作电位,放电模式可被用于味觉特征的编码。而且味觉感受细胞和味觉神经纤维的响应有很多相似点,表明味觉感受细胞产生的动作电位可能是味觉信息传递到味觉神经纤维的主要组成部分。其电活动对于味觉信息的前期处理起主导作用。对味觉感受细胞离子通道的研究能够在分子水平上揭示味觉感受细胞兴奋性的功能和机制。味觉感受细胞膜上存在多种电压门控离子通道。实验证据表明电压门控离子通道作为味觉感受细胞兴奋性的分子基础,在味觉信息的前期编码和处理中具有重要意义。 味觉感受细胞对于生理味质刺激的响应可借助全细胞膜片钳、细胞粘附式膜片钳技术和基于膜片钳的胞外记录技术进行记录。然而由于受这些实验技术的限制,目前对于味觉物质刺激下单个味觉感受细胞产生的动作电位及其离子机制、放电模式以及它所携带的味觉信息了解甚少。这些事实提示,对细胞和亚细胞水平上的前期味觉功能及其机理的研究需要结合理论和实验等多种手段,包括计算机建模和仿真技术和仿生细胞传感器,多角度协同进行研究。本文基于膜片钳全细胞记录以及单通道记录数据,对味觉细胞的配体门控通道和电压门控通道的动力学特性和机理进行描述分析,从而对这个味觉细胞数学模型进行建立。仿真在酸味刺激过程中的时域放电模式。对酸敏感机理以及放电特性进行分析。 随着微电子微机械加工技术的发展,利用光电技术的优点,采用光寻址电位传感器(LAPS, Light Addressable Potentiometric Sensor)芯片作为味觉感受和放电响应研究的平台。激光器在LAPS表面扫描寻址,即可对想要检测点的表面电位进行记录,大大提高了细胞传感器的灵活性和可行性。克服了味觉细胞培养密度低的缺点。可用于对味觉细胞在味质刺激下产生的电信号和化学信号检测。 本论文的主要内容和贡献如下： 1.通过实验建立了味觉酸通道PKD2L1/1L3的动力学模型,通过理论分析该通道OFF响应产生的机理,发现了味觉酸通道具有碱激活工作方式,并具有酸依赖抑制和钙依赖抑制特性。此外,确定了在酸性条件下该通道活动处于抑制状态； 2.建立了PKD2通道的单通道模型,并用模型仿真发现了该通道在酸洗脱后具有OFF响应特性。通过膜片钳实验发现单转PKD2L1时在酸刺激洗脱后会产生OFF响应。从模型理论和实验中得到了论证。通过该模型的分析与实验论证,进一步支持了PKD1L3/2L1通道的OFF响应特性是碱激活机理产生的； 3.首次建立了酸敏感味觉的全细胞电生理数学模型,仿真发现了在酸敏感中,味觉细胞有ON和OFF放电响应,且存在的三种可能性,通过微电极阵列芯片进行了实验验证。此外,通过该模型仿真分析得到了PKDL通道的钙依赖抑制特性在酸敏感中起着负反馈作用的结果； 4.设计了一种基于LAPS的味觉仿生单细胞传感器,培养在芯片表面的味觉细胞存在功能性的Ⅱ型和Ⅲ型。且通过味觉细胞模型和细胞与芯片耦合模型,仿真了味觉细胞在酸味刺激下的胞外信号。实验结果表明：味觉信息是能在初级味觉敏感系统中编码的,且ATP对Ⅲ型味觉细胞具有加强和抑制的双重作用。并结合免疫组化荧光检测方法对LAPS芯片表面培养的味觉细胞进行特异性标记,表明仿生味觉单细胞传感器能够对外界环境离子或是分子的改变进行检测。 5.提出了基于LAPS芯片的光寻址特性一种新的仿生味觉细胞网络的传感器,用于检测神经递质5-HT,并应用于味觉细胞间的信息通讯和信息传递的研究。该传感器对5-HT的最低检测限为3×10-13 M,检测灵敏度为19.1mVp5-HT。在酸味或苦甜鲜混合味质的刺激下,舌上皮组织和味觉细胞释放的5-HT局部浓度均为nM级。同时利用荧光共标记手段,对培养的味觉细胞群进行特异性标记,表明离体培养的味觉细胞群中同时具有功能性的Ⅱ型和Ⅲ型细胞存在。
[Abstract]:Taste sensitive cells are the basic units of taste sensitivity, and realize the important pre information processing functions such as chemical electrical signal conversion in the taste sense. The study shows that acid, sweet, bitter, salty and tasty flavor can stimulate the action potential of taste sensory cells, and the mode of discharge can be used in the coding of taste sensation. The action potential produced by the taste receptive cells may be the main component of the taste information transferred to the taste nerve fibers. The electrical activity plays a leading role in the preprocessing of the taste information. The study of the ion channels of the taste sensory cells can reveal the taste sensory cells at the molecular level. There are many voltage gated ion channels on the taste receptive cell membrane. Experimental evidence shows that the voltage gated ion channel is the molecular basis of the excitatory of the taste receptive cells and is of great significance in the pre coding and processing of the taste information.
The response of taste receptors to physiological stimuli can be recorded by whole cell patch clamp, cell adhesion patch clamp technique and extracellular recording technique based on patch clamp. However, due to the restriction of these experimental techniques, the action potential and the ionic machine produced by the taste substances under the stimulation of a single taste sensory cell are present. These facts suggest that the study of the prophase taste function and its mechanism at the cellular and subcellular level and its mechanism need to be combined with a variety of means such as theory and experiment, including computer modeling and simulation techniques and biomimetic cell sensors, and multi angle collaboration. The whole cell recording of patch clamp and the single channel recording data are used to describe the dynamic characteristics and mechanism of the ligand gated and voltage gated channels of the gustatory cells, so as to establish the mathematical model of the taste cells. Line analysis.
With the development of microelectronic micromachining technology and the advantage of the photoelectric technology, the LAPS (Light Addressable Potentiometric Sensor) chip is used as a platform for the research of taste sensation and discharge response. The surface potential of the desired detection point can be recorded by the laser scanning on the surface of the LAPS. It improves the flexibility and feasibility of cell sensors. It overcomes the disadvantages of low density of the culture of taste cells. It can be used to detect the electrical signals and chemical signals produced by the taste cells under the taste stimulation.
The main contents and contributions of this paper are as follows:
1. the kinetic model of the taste acid channel PKD2L1/1L3 was established through the experiment. Through the theoretical analysis of the mechanism of the channel OFF response, it was found that the taste acid channel has alkali activation working mode, and it has the inhibition of acid dependence and calcium dependence. In addition, it is determined that the channel activity is in the inhibition state under the acidic condition.
2. a single channel model of PKD2 channel is established and the model simulation is used to find that the channel has the response characteristic of OFF after pickling. Through the patch clamp experiment, it is found that the response of OFF after the acid stimulus is eluted by the acid stimulation. The model theory and experiment are demonstrated. Through the analysis and experimental demonstration of the model, the PKD is further supported by the PKD. The OFF response characteristic of 1L3/2L1 channels is generated by alkali activation mechanism.
3. the whole cell electrophysiological mathematical model of acid sensitive taste was established for the first time. The simulation found that in acid sensitive, the taste cells had ON and OFF discharge responses, and the three possibilities were verified by microelectrode array chips. In addition, the simulation analysis of the model showed that the calcium dependence of PKDL channel was sensitive to acid sensitivity. As a result of negative feedback.
4. the taste bionic single cell sensor based on LAPS was designed to cultivate the functional type II and type III of the taste cells on the surface of the chip. Through the model of the taste cell and the cell and chip, the extracellular signal of the taste cells under the acid stimulation was simulated. The experimental results show that the taste information can be in the primary taste sense. In the sensitive system, the ATP has double effects on the type III taste cells, and the specific labeling of the taste cells cultured on the surface of LAPS chips by the immunohistochemical fluorescence detection method indicates that the biomimetic mono cell sensor can detect the changes in the external environment and the molecular changes.
5. a new bionic taste cell network sensor based on LAPS chip is proposed, which is used to detect neurotransmitter 5-HT and apply to the study of information communication and information transmission between taste cells. The minimum detection limit for 5-HT is 3 x 10-13 M, and the sensitivity is 19.1mVp5-HT. in sour or bitter sweet and fresh mixture Under the stimulation of flavor, the local concentration of 5-HT released by the tongue epithelium and the taste cells were all nM. The specific labeling of the cultured taste cell groups was carried out by the means of fluorescence co labeling, indicating the existence of functional type II and type III cells in the gustatory cell group in vitro culture.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R314

【参考文献】

中国期刊全文数据库 前1条

1 许改霞,吴一聪,李蓉,王平,严伟民,郑筱祥;细胞传感器的研究进展[J];科学通报;2002年15期

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本文编号：1977624