本文選題：味覺(jué)細(xì)胞電生理模型 + PKD1L3/2L1通道�。� 參考：《浙江大學(xué)》2010年博士論文

【摘要】： 味覺(jué)感受細(xì)胞是味覺(jué)敏感的基本單元,實(shí)現(xiàn)味覺(jué)中化學(xué)-電信號(hào)轉(zhuǎn)換等重要的前期信息處理功能。研究表明酸、甜、苦、咸和鮮味味質(zhì)能刺激味覺(jué)感受細(xì)胞產(chǎn)生動(dòng)作電位,放電模式可被用于味覺(jué)特征的編碼。而且味覺(jué)感受細(xì)胞和味覺(jué)神經(jīng)纖維的響應(yīng)有很多相似點(diǎn),表明味覺(jué)感受細(xì)胞產(chǎn)生的動(dòng)作電位可能是味覺(jué)信息傳遞到味覺(jué)神經(jīng)纖維的主要組成部分。其電活動(dòng)對(duì)于味覺(jué)信息的前期處理起主導(dǎo)作用。對(duì)味覺(jué)感受細(xì)胞離子通道的研究能夠在分子水平上揭示味覺(jué)感受細(xì)胞興奮性的功能和機(jī)制。味覺(jué)感受細(xì)胞膜上存在多種電壓門控離子通道。實(shí)驗(yàn)證據(jù)表明電壓門控離子通道作為味覺(jué)感受細(xì)胞興奮性的分子基礎(chǔ),在味覺(jué)信息的前期編碼和處理中具有重要意義。 味覺(jué)感受細(xì)胞對(duì)于生理味質(zhì)刺激的響應(yīng)可借助全細(xì)胞膜片鉗、細(xì)胞粘附式膜片鉗技術(shù)和基于膜片鉗的胞外記錄技術(shù)進(jìn)行記錄。然而由于受這些實(shí)驗(yàn)技術(shù)的限制,目前對(duì)于味覺(jué)物質(zhì)刺激下單個(gè)味覺(jué)感受細(xì)胞產(chǎn)生的動(dòng)作電位及其離子機(jī)制、放電模式以及它所攜帶的味覺(jué)信息了解甚少。這些事實(shí)提示,對(duì)細(xì)胞和亞細(xì)胞水平上的前期味覺(jué)功能及其機(jī)理的研究需要結(jié)合理論和實(shí)驗(yàn)等多種手段,包括計(jì)算機(jī)建模和仿真技術(shù)和仿生細(xì)胞傳感器,多角度協(xié)同進(jìn)行研究。本文基于膜片鉗全細(xì)胞記錄以及單通道記錄數(shù)據(jù),對(duì)味覺(jué)細(xì)胞的配體門控通道和電壓門控通道的動(dòng)力學(xué)特性和機(jī)理進(jìn)行描述分析,從而對(duì)這個(gè)味覺(jué)細(xì)胞數(shù)學(xué)模型進(jìn)行建立。仿真在酸味刺激過(guò)程中的時(shí)域放電模式。對(duì)酸敏感機(jī)理以及放電特性進(jìn)行分析。 隨著微電子微機(jī)械加工技術(shù)的發(fā)展,利用光電技術(shù)的優(yōu)點(diǎn),采用光尋址電位傳感器(LAPS, Light Addressable Potentiometric Sensor)芯片作為味覺(jué)感受和放電響應(yīng)研究的平臺(tái)。激光器在LAPS表面掃描尋址,即可對(duì)想要檢測(cè)點(diǎn)的表面電位進(jìn)行記錄,大大提高了細(xì)胞傳感器的靈活性和可行性。克服了味覺(jué)細(xì)胞培養(yǎng)密度低的缺點(diǎn)。可用于對(duì)味覺(jué)細(xì)胞在味質(zhì)刺激下產(chǎn)生的電信號(hào)和化學(xué)信號(hào)檢測(cè)。 本論文的主要內(nèi)容和貢獻(xiàn)如下： 1.通過(guò)實(shí)驗(yàn)建立了味覺(jué)酸通道PKD2L1/1L3的動(dòng)力學(xué)模型,通過(guò)理論分析該通道OFF響應(yīng)產(chǎn)生的機(jī)理,發(fā)現(xiàn)了味覺(jué)酸通道具有堿激活工作方式,并具有酸依賴抑制和鈣依賴抑制特性。此外,確定了在酸性條件下該通道活動(dòng)處于抑制狀態(tài)； 2.建立了PKD2通道的單通道模型,并用模型仿真發(fā)現(xiàn)了該通道在酸洗脫后具有OFF響應(yīng)特性。通過(guò)膜片鉗實(shí)驗(yàn)發(fā)現(xiàn)單轉(zhuǎn)PKD2L1時(shí)在酸刺激洗脫后會(huì)產(chǎn)生OFF響應(yīng)。從模型理論和實(shí)驗(yàn)中得到了論證。通過(guò)該模型的分析與實(shí)驗(yàn)論證,進(jìn)一步支持了PKD1L3/2L1通道的OFF響應(yīng)特性是堿激活機(jī)理產(chǎn)生的； 3.首次建立了酸敏感味覺(jué)的全細(xì)胞電生理數(shù)學(xué)模型,仿真發(fā)現(xiàn)了在酸敏感中,味覺(jué)細(xì)胞有ON和OFF放電響應(yīng),且存在的三種可能性,通過(guò)微電極陣列芯片進(jìn)行了實(shí)驗(yàn)驗(yàn)證。此外,通過(guò)該模型仿真分析得到了PKDL通道的鈣依賴抑制特性在酸敏感中起著負(fù)反饋?zhàn)饔玫慕Y(jié)果； 4.設(shè)計(jì)了一種基于LAPS的味覺(jué)仿生單細(xì)胞傳感器,培養(yǎng)在芯片表面的味覺(jué)細(xì)胞存在功能性的Ⅱ型和Ⅲ型。且通過(guò)味覺(jué)細(xì)胞模型和細(xì)胞與芯片耦合模型,仿真了味覺(jué)細(xì)胞在酸味刺激下的胞外信號(hào)。實(shí)驗(yàn)結(jié)果表明：味覺(jué)信息是能在初級(jí)味覺(jué)敏感系統(tǒng)中編碼的,且ATP對(duì)Ⅲ型味覺(jué)細(xì)胞具有加強(qiáng)和抑制的雙重作用。并結(jié)合免疫組化熒光檢測(cè)方法對(duì)LAPS芯片表面培養(yǎng)的味覺(jué)細(xì)胞進(jìn)行特異性標(biāo)記,表明仿生味覺(jué)單細(xì)胞傳感器能夠?qū)ν饨绛h(huán)境離子或是分子的改變進(jìn)行檢測(cè)。 5.提出了基于LAPS芯片的光尋址特性一種新的仿生味覺(jué)細(xì)胞網(wǎng)絡(luò)的傳感器,用于檢測(cè)神經(jīng)遞質(zhì)5-HT,并應(yīng)用于味覺(jué)細(xì)胞間的信息通訊和信息傳遞的研究。該傳感器對(duì)5-HT的最低檢測(cè)限為3×10-13 M,檢測(cè)靈敏度為19.1mVp5-HT。在酸味或苦甜鮮混合味質(zhì)的刺激下,舌上皮組織和味覺(jué)細(xì)胞釋放的5-HT局部濃度均為nM級(jí)。同時(shí)利用熒光共標(biāo)記手段,對(duì)培養(yǎng)的味覺(jué)細(xì)胞群進(jìn)行特異性標(biāo)記,表明離體培養(yǎng)的味覺(jué)細(xì)胞群中同時(shí)具有功能性的Ⅱ型和Ⅲ型細(xì)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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R314

【參考文獻(xiàn)】

中國(guó)期刊全文數(shù)據(jù)庫(kù) 前1條

1 許改霞,吳一聰,李蓉,王平,嚴(yán)偉民,鄭筱祥;細(xì)胞傳感器的研究進(jìn)展[J];科學(xué)通報(bào);2002年15期

，

本文編號(hào)：1977624