味觉细胞芯片及其在味觉传导机理中的研究

发布时间：2018-05-19 09:46

本文选题：味觉 + 细胞芯片　；参考：《浙江大学》2006年硕士论文

【摘要】：味觉作为人类基本感觉之一,其研究却远远落后于视觉、听觉等。不仅如此,目前的味觉电生理记录,仍主要依赖于以膜片钳为代表的细胞膜微电极穿刺或钳制技术。该类方法因其对细胞的穿刺损害作用,难以实现长时程测量,同时也难以实现对细胞网络的多位点同时测量。因此,研究者们开发出基于微电极阵列(Microelectrode Array,MEA)、场效应管(Field Effect Transistor,FET)阵列以及光寻址电位传感器(Light Addressable Potentiometric Sensor,LAPS)的细胞芯片,其实质就是在芯片表面培养可兴奋性细胞如神经元等,细胞通过一层薄的电解液同芯片的电极或栅极相耦合,从而芯片可以实时监测药物或者电刺激下细胞的电生理变化。细胞芯片可对多个细胞同时进行长期、无损的胞外电信号测量,已在细胞电生理研究、药物筛选以及环境监测等生物医学领域得到了广泛应用。本文结合我们实验室多年来在人工味觉和细胞芯片方面的研究,提出了基于LAPS的味觉细胞芯片。味觉受体细胞具有神经细胞特性,也属于可兴奋性细胞,将其培养在LAPS芯片表面,味觉细胞电位的改变即可通过检测LAPS的光生电流得以测量。与MEA和FET阵列芯片相比,LAPS芯片不仅制各工艺简单,而且其光源可移动寻址,方便对芯片表面随机培养的细胞进行跟踪定位,克服了MEA和FET阵列芯片固定检测位点几何特性的限制。采用微机械加工技术,我们制备了LAPS味觉细胞芯片,并用多聚鸟氨酸和层粘素混合的磷酸缓冲液处理以增强细胞在芯片表面的黏附性。采用微吸管吸取技术,我们体外分离出大鼠的味蕾,将其培养在LAPS芯片上。选取去甲肾上腺素和河豚毒素作为刺激药物,作用于芯片上已贴壁生长的味觉细胞,验证了LAPS芯片检测味觉细胞动作电位变化的可行性。然后,选择四种基本味质(NaCl,HCl,蔗糖,MgSO_4)作用于味觉细胞,检测LAPS的响应。采用LabVIEW采集数据、Matlab进行时域和频域分析,我们定性分析了不同味质作用于味觉细胞后胞外电信号的变化,证实了大鼠菌状乳头的味觉细胞对于咸味和酸味的敏感性,实现了细胞芯片在味觉传导机理研究中的应用。
[Abstract]:Taste sense as one of the basic human senses, its research lags far behind the visual, auditory, and so on. Moreover, the current taste electrophysiological records still rely mainly on the membrane microelectrode puncture or clamp technology represented by patch clamp. Because of its damage to cell puncture, this method is difficult to realize long-term measurement and multi-site simultaneous measurement of cell network. So researchers have developed a cell chip based on microelectrode arrays, Field Effect transistorization arrays and Light Addressable Potentiometric sensors (LAPs), which essentially grow excitable cells, such as neurons, on the surface of the chip. The cell is coupled with the electrode or grid of the chip through a thin layer of electrolyte, so that the chip can monitor the electrophysiological changes of the cell in real time under drug or electrical stimulation. Cell chips can be used to measure long-term, non-destructive extracellular electrical signals on many cells simultaneously, and have been widely used in cell electrophysiology research, drug screening and environmental monitoring and other biomedical fields. In this paper, a taste cell chip based on LAPS is proposed based on the research of artificial taste and cell chip in our laboratory for many years. Taste receptor cells are neuronal and excitable cells. When cultured on LAPS microarray, the changes of taste cell potential can be measured by detecting photogenerated currents of LAPS. Compared with MEA and FET chips, laps chip is not only easy to manufacture, but also its light source can be moved and addressable, which makes it easy to track and locate the cells cultured randomly on the surface of the chip. It overcomes the limitation of geometric characteristics of fixed detection sites in MEA and FET array chips. A LAPS taste cell chip was prepared by micromachining and was treated with polyornithine and laminin phosphate buffer to enhance cell adhesion on the chip surface. The taste buds of rats were isolated by micropipette extraction and cultured on LAPS microarray. Norepinephrine and tetrodotoxin were selected as stimulators to act on the taste cells grown on the chip. The feasibility of detecting the action potential of taste cells by LAPS chip was verified. Then, four basic flavors, NaCl-HCl, Sucrose Mg-SO4) were selected to act on the taste cells to detect the response of LAPS. Time domain and frequency domain analysis were carried out with LabVIEW data. We qualitatively analyzed the changes of extracellular electrical signals after different taste substances acted on taste cells, and confirmed the sensitivity of taste cells of rat papilla to salty and sour taste. The application of cell chip in the study of taste conduction mechanism is realized.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R33

【引证文献】