【摘要】：細胞膜是細胞進行物質(zhì)交換和信息傳遞的重要通道，細胞在接受刺激后，眾多的信號通路同時在細胞膜上發(fā)生，細胞膜上必然存在著協(xié)調(diào)不同通路轉(zhuǎn)導(dǎo)的區(qū)域。脂筏就是參與信號轉(zhuǎn)導(dǎo)的平臺之一。脂筏是細胞膜上富含膽固醇和鞘磷脂的微結(jié)構(gòu)域，與膜的信號轉(zhuǎn)導(dǎo)以及蛋白質(zhì)分選均有密切關(guān)系。脂筏中的小窩區(qū)域是一個去污劑不溶性膜區(qū)域，以存在小窩蛋白分子（caveolin）為特征，可參與細胞膽固醇運輸，細胞膜合成，信號傳導(dǎo)和腫瘤生成等多種細胞生命活動。目前已知的小窩蛋白有三類：caveolin-1（包括兩種變異體α和β），，caveolin-2和caveolin-3。研究表明，caveolin-1具有與膽固醇結(jié)合能力，并參與了細胞內(nèi)膽固醇穩(wěn)態(tài)的維持和膽固醇的轉(zhuǎn)運過程。甲基-β-環(huán)糊精(M-β-CD)是一種去污劑，能夠?qū)⒓毎ど系哪懝檀紡募毎こ樘岢鰜�，從而破壞脂筏的完整性�?細胞膜電位是由細胞膜兩側(cè)存在的離子濃度差和細胞膜對離子的通透性（離子通道）不同而形成的。不同離子通道在細胞膜上的表達和功能的不同構(gòu)成了復(fù)雜而又規(guī)律的細胞電活動�？缂毎さ碾娀顒涌梢杂绊懺S多細胞活動及功能。在細胞膜有不同的感覺跨膜電活動的蛋白分子，這些分子除顯而易見的離子通道蛋白分子外，還有其他一些非生電蛋白分子例如一些酶類。 在之前的研究中發(fā)現(xiàn)，在卵母細胞中細胞膜去極化可導(dǎo)致PIP2水平升高，這依賴于PKC、PI4K和PLC等酶活性的增強，在這些酶的參與下，磷脂酰肌醇（PIP2）代謝加快并在細胞膜上維持高水平狀態(tài)，可以使卵母細胞中表達的、功能依賴于PIP2的KCNQ2/Q3鉀電流增大。 本課題研究開始于我們觀察到高滲外液能阻斷細胞膜去極化增大KCNQ2/Q3通道電流的作用。細胞膜去極化導(dǎo)致的細胞膜PIP2合成代謝增加、KCNQ2/Q3功能增強的機制可能與高滲外液改變細胞膜物理性狀而影響細胞功能的機制類似。因此，我們針對細胞膜完整性，具體是脂筏在細胞膜電位調(diào)節(jié)PIP2代謝中的作用及機制展開研究。具體研究內(nèi)容如下： 目的：研究甲基-β-環(huán)糊精（M-β-CD）對KCNQ2/Q3電流的影響，由此推測M-β-CD對細胞膜PIP2代謝的影響并對其分子機制進行探索研究；了解細胞膜脂筏在細胞膜電位調(diào)節(jié)PIP2代謝中作用及分子機制。 方法：體外轉(zhuǎn)錄的方法制備KCNQ2和KCNQ3通道的cRNA并使之表達于爪蟾卵母細胞；使用雙電極電壓鉗記錄表達在卵母細胞上的KCNQ2/Q3電流，并觀察不同實驗條件下電流的變化；Western blot檢測不同處理組卵母細胞中PI4K和Caveolin-1表達情況。 結(jié)果：(1) M-β-CD對表達于卵母細胞的KCNQ2/Q3電流有很強的增大作用，其無效類似物α-CD沒有增加KCNQ2/Q3電流；(2)PLC阻斷劑U73122，PI4K阻斷劑PIK93，PKC阻斷劑Bis均能阻斷M-β-CD增大KCNQ2/Q3電流的的作用；(3) M-β-CD能完全阻斷細胞膜去極化以及PMA對KCNQ2/Q3電流的增大作用；(4)NaCl及糖類高滲外液能阻斷PMA以及M-β-CD對KCNQ2/Q3電流的增大作用，且對KCNQ2/Q3電流有輕微抑制；(5)細胞外高濃度膽固醇能阻斷M-β-CD對KCNQ2/Q3電流的增大作用，且對KCNQ2/Q3電流有強的抑制作用；(6)Western blot結(jié)果顯示，M-β-CD處理組細胞中PI4K含量顯著高于正常對照組，而細胞中Caveolin-1比例顯著減少。細胞膜去極化處理組細胞中PI4K含量顯著增多但Caveolin-1比例與對照組無顯著性差異。 結(jié)論：實驗結(jié)果表明M-β-CD明顯增大表達在卵母細胞上的KCNQ2/Q3電流，表明M-β-CD使卵母細胞膜PIP2含量增加。細胞膜去極化導(dǎo)致的細胞膜PIP2合成代謝增加、KCNQ2/Q3功能增強可能與M-β-CD作用的機制類似。細胞膜脂筏結(jié)構(gòu)的完整性可能在細胞膜去極化導(dǎo)致的細胞膜PIP2合成代謝的作用中發(fā)揮重要作用。
[Abstract]:Cell membrane is an important channel for substance exchange and information transmission. After stimulation, many signal pathways occur on the cell membrane at the same time. There must be areas on the cell membrane that coordinate different pathways. Lipid raft is one of the platforms involved in signal transduction. Lipid raft is rich in cholesterol and sphingomyelin on the cell membrane. Microstructure domains are closely related to membrane signal transduction and protein sorting. The litter region in the lipid raft is a detergent-insoluble membrane region characterized by caveolin molecules, which can participate in cell cholesterol transport, cell membrane synthesis, signal transduction and tumor formation. Caveolin-1 (including two variants alpha and beta), Caveolin-2 and caveolin-3. Studies have shown that caveolin-1 binds to cholesterol and is involved in the maintenance of intracellular cholesterol homeostasis and cholesterol transport. Methyl-beta-cyclodextrin (M-beta-CD) is a detergent that removes cholesterol from cell membranes. The cell membrane is extracted to destroy the integrity of lipid rafts.
Membrane potential is formed by the difference of ion concentration on both sides of the cell membrane and the difference of ion permeability (ion channel) between the two sides of the cell membrane. Cell membranes have different protein molecules that sense transmembrane electrical activity. These molecules, in addition to the obvious ion channel proteins, have other non-electrogenic proteins such as enzymes.
Previous studies have shown that depolarization of cell membrane in oocytes leads to an increase in PIP2 levels, which is dependent on enzymatic activities such as PKC, PI4K and PLC. With the participation of these enzymes, phosphoinositide (PIP2) metabolism is accelerated and maintains a high level on the cell membrane, enabling oocytes to express and function dependent on PIP2 KCNQ2/Q. 3 potassium current increases.
This study began with our observation that hypertonic exudates could block the depolarization of cell membranes and increase the current of KCNQ2/Q3 channels. The depolarization of cell membranes resulted in the increase of PIP2 synthesis and metabolism. The mechanism of KCNQ2/Q3 enhancement may be similar to that of hypertonic exudates which altered the physical properties of cell membranes and affected cell functions. The role and mechanism of lipid rafts in the regulation of PIP2 metabolism by membrane potential were studied.
AIM: To study the effect of methyl-beta-cyclodextrin (M-beta-CD) on KCNQ2/Q3 current, and to speculate the effect of M-beta-CD on the metabolism of cell membrane PIP2 and explore its molecular mechanism.
Methods: KCNQ2 and KCNQ3 channel cRNA were transcribed in vitro and expressed in Xenopus oocytes. The KCNQ2/Q3 currents were recorded by two-electrode voltage clamp, and the changes of current under different experimental conditions were observed. The expressions of PI4K and Caveolin-1 were detected by Western blot.
Results: (1) M-beta-CD had a strong effect on KCNQ2/Q3 currents in oocytes, but its ineffective analogue alpha-CD did not increase KCNQ2/Q3 currents; (2) PLC blocker U73122, PI4K blocker PIK93 and PKC blocker Bis could block the effect of M-beta-CD on KCNQ2/Q3 currents; (3) M-beta-CD completely blocked cell membrane depolarization and P-Q3 currents. MA can increase KCNQ2/Q3 current; (4) NaCl and carbohydrate hypertonic solution can block the increase of PMA and M-beta-CD on KCNQ2/Q3 current, and slightly inhibit KCNQ2/Q3 current; (5) extracellular high concentration of cholesterol can block the increase of M-beta-CD on KCNQ2/Q3 current, and has a strong inhibition on KCNQ2/Q3 current; (6) Western blot The results showed that the content of PI4K in M-beta-CD treated cells was significantly higher than that in the normal control group, while the proportion of Caveolin-1 in cells was significantly decreased.
CONCLUSION: The results showed that M-beta-CD significantly increased KCNQ2/Q3 currents on oocytes, indicating that M-beta-CD increased the content of membrane PIP2, increased the synthesis and metabolism of membrane PIP2 induced by cell membrane depolarization, and the enhancement of KCNQ2/Q3 function may be similar to the mechanism of M-beta-CD. The integrity of membrane lipid raft structure may be fine. Membrane depolarization plays an important role in the role of cell membrane PIP2 metabolism.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R96

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1 張熙東;甲基-β-環(huán)糊精及脂筏對細胞膜磷脂PIP2代謝的影響及分子機制研究[D];河北醫(yī)科大學(xué);2014年

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