發(fā)布時間：2018-06-04 14:01

  本文選題：味覺細胞電生理模型 + PKD1L3/2L1通道��； 參考：《浙江大學》2010年博士論文

【摘要】： 味覺感受細胞是味覺敏感的基本單元,實現(xiàn)味覺中化學-電信號轉(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)能刺激味覺感受細胞產(chǎn)生動作電位,放電模式可被用于味覺特征的編碼。而且味覺感受細胞和味覺神經(jīng)纖維的響應有很多相似點,表明味覺感受細胞產(chǎn)生的動作電位可能是味覺信息傳遞到味覺神經(jīng)纖維的主要組成部分。其電活動對于味覺信息的前期處理起主導作用。對味覺感受細胞離子通道的研究能夠在分子水平上揭示味覺感受細胞興奮性的功能和機制。味覺感受細胞膜上存在多種電壓門控離子通道。實驗證據(jù)表明電壓門控離子通道作為味覺感受細胞興奮性的分子基礎,在味覺信息的前期編碼和處理中具有重要意義。 味覺感受細胞對于生理味質(zhì)刺激的響應可借助全細胞膜片鉗、細胞粘附式膜片鉗技術和基于膜片鉗的胞外記錄技術進行記錄。然而由于受這些實驗技術的限制,目前對于味覺物質(zhì)刺激下單個味覺感受細胞產(chǎn)生的動作電位及其離子機制、放電模式以及它所攜帶的味覺信息了解甚少。這些事實提示,對細胞和亞細胞水平上的前期味覺功能及其機理的研究需要結合理論和實驗等多種手段,包括計算機建模和仿真技術和仿生細胞傳感器,多角度協(xié)同進行研究。本文基于膜片鉗全細胞記錄以及單通道記錄數(shù)據(jù),對味覺細胞的配體門控通道和電壓門控通道的動力學特性和機理進行描述分析,從而對這個味覺細胞數(shù)學模型進行建立。仿真在酸味刺激過程中的時域放電模式。對酸敏感機理以及放電特性進行分析。 隨著微電子微機械加工技術的發(fā)展,利用光電技術的優(yōu)點,采用光尋址電位傳感器(LAPS, Light Addressable Potentiometric Sensor)芯片作為味覺感受和放電響應研究的平臺。激光器在LAPS表面掃描尋址,即可對想要檢測點的表面電位進行記錄,大大提高了細胞傳感器的靈活性和可行性。克服了味覺細胞培養(yǎng)密度低的缺點�？捎糜趯ξ队X細胞在味質(zhì)刺激下產(chǎn)生的電信號和化學信號檢測。 本論文的主要內(nèi)容和貢獻如下： 1.通過實驗建立了味覺酸通道PKD2L1/1L3的動力學模型,通過理論分析該通道OFF響應產(chǎn)生的機理,發(fā)現(xiàn)了味覺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響應特性。通過膜片鉗實驗發(fā)現(xiàn)單轉(zhuǎn)PKD2L1時在酸刺激洗脫后會產(chǎn)生OFF響應。從模型理論和實驗中得到了論證。通過該模型的分析與實驗論證,進一步支持了PKD1L3/2L1通道的OFF響應特性是堿激活機理產(chǎn)生的； 3.首次建立了酸敏感味覺的全細胞電生理數(shù)學模型,仿真發(fā)現(xiàn)了在酸敏感中,味覺細胞有ON和OFF放電響應,且存在的三種可能性,通過微電極陣列芯片進行了實驗驗證。此外,通過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反饋作用的結果； 4.設計了一種基于LAPS的味覺仿生單細胞傳感器,培養(yǎng)在芯片表面的味覺細胞存在功能性的Ⅱ型和Ⅲ型。且通過味覺細胞模型和細胞與芯片耦合模型,仿真了味覺細胞在酸味刺激下的胞外信號。實驗結果表明：味覺信息是能在初級味覺敏感系統(tǒng)中編碼的,且ATP對Ⅲ型味覺細胞具有加強和抑制的雙重作用。并結合免疫組化熒光檢測方法對LAPS芯片表面培養(yǎng)的味覺細胞進行特異性標記,表明仿生味覺單細胞傳感器能夠?qū)ν饨绛h(huán)境離子或是分子的改變進行檢測。 5.提出了基于LAPS芯片的光尋址特性一種新的仿生味覺細胞網(wǎng)絡的傳感器,用于檢測神經(jīng)遞質(zhì)5-HT,并應用于味覺細胞間的信息通訊和信息傳遞的研究。該傳感器對5-HT的最低檢測限為3×10-13 M,檢測靈敏度為19.1mVp5-HT。在酸味或苦甜鮮混合味質(zhì)的刺激下,舌上皮組織和味覺細胞釋放的5-HT局部濃度均為nM級。同時利用熒光共標記手段,對培養(yǎng)的味覺細胞群進行特異性標記,表明離體培養(yǎng)的味覺細胞群中同時具有功能性的Ⅱ型和Ⅲ型細胞存在。
[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.
【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2010
【分類號】：R314

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中國期刊全文數(shù)據(jù)庫 前1條

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本文編號：1977624