本文關(guān)鍵詞： 脂筏 頸上神經(jīng)節(jié) M_1受體 B_2受體 M電流 信號(hào)分子 甲基-β-環(huán)糊精　出處：《河北醫(yī)科大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：M電流是廣泛存在于神經(jīng)系統(tǒng)的一種鉀電流,是影響神經(jīng)元興奮性的主要機(jī)制之一,是唯一在神經(jīng)元閾電位附近激活的電流,它的功能降低可以引起神經(jīng)元興奮性增高,誘發(fā)癲癇等疾病。眾多神經(jīng)遞質(zhì)和神經(jīng)肽可以調(diào)節(jié)M電流進(jìn)而影響神經(jīng)元興奮性。近年來(lái),對(duì)神經(jīng)遞質(zhì)等調(diào)節(jié)M電流的分子機(jī)制有了很大進(jìn)展,其中以乙酰膽堿激活M型膽堿受體(M1)和緩激肽激活其II型緩激肽受體(B2受體)后調(diào)節(jié)M電流的分子機(jī)制最有代表性。雖然兩種受體激活都可以抑制M電流,但其分子機(jī)制有所不同,其中對(duì)細(xì)胞內(nèi)Ca2+濃度影響的不同是關(guān)鍵之處。然而,對(duì)于造成這一不同的進(jìn)一步機(jī)制則有待進(jìn)一步研究。本實(shí)驗(yàn)試圖從細(xì)胞膜脂筏(lipid raft)的角度來(lái)解釋不同膜受體介導(dǎo)的細(xì)胞信號(hào)通路對(duì)M電流調(diào)節(jié)不同的機(jī)制。 脂筏是細(xì)胞膜上的一種脂質(zhì)微區(qū),在大多數(shù)哺乳動(dòng)物的細(xì)胞膜上都有分布,一般大小為55-300 nm,富含膽固醇和鞘磷脂,具有低浮力密度和不溶于去污劑的特性。近期研究表明,脂筏在信號(hào)轉(zhuǎn)導(dǎo)事件中發(fā)揮著重要的作用;脂筏中特異聚集的不同信號(hào)分子可能決定了不同信號(hào)轉(zhuǎn)導(dǎo)通路的特異性。本研究將以與M1受體、B2受體以及所相關(guān)的細(xì)胞信號(hào)分子在脂筏中分布的異同為中心,觀察脂筏在M電流的特異性調(diào)節(jié)中的作用。 目的:研究大鼠頸上神經(jīng)節(jié)神經(jīng)元細(xì)胞膜脂筏在膜受體特異調(diào)節(jié)M電流中的作用 方法: 1脂筏提取及脂筏中膜蛋白的鑒定 1.1蔗糖密度梯度離心法分離脂筏及脂筏的鑒定 摘取40只7天SD大鼠SCG,用玻璃勻漿器進(jìn)行勻漿,離心5000 rpm,5 min。取上清。將約1.5 ml上清置于8.9 ml離心管底部,加入80%蔗糖1.5 ml,輕輕混勻避免氣泡的產(chǎn)生,在液面上方緩慢加入3 ml 35%蔗糖,同法操作,再加入約3 ml 5%蔗糖,制備40%/35%/5%的蔗糖梯度。39,000 rpm,4℃,離心21 h。將離心后的液體從上到下連續(xù)取出九個(gè)部分,每部分約一毫升。從每個(gè)部分中取出4μl,均勻地點(diǎn)于尼龍膜上,進(jìn)行Dot blots實(shí)驗(yàn),利用霍亂毒素B測(cè)定神經(jīng)節(jié)苷脂GM1在九個(gè)部分的分布情況,而GM1是脂筏的標(biāo)記分子之一。 1.2去污劑法分離脂筏 摘取10只7天SD大鼠SCG,用玻璃勻漿器勻漿,離心5,000 rpm,10 min,取上清,40,200 rpm, 4℃,1 h。沉淀用200μl含500 mM Na2CO3及1% triton低滲勻漿Buffer懸浮并超聲,冰上靜置30 min,15,000 g, 4℃,30 min,分成去污劑溶解與不溶兩種組分,然后用5×SDS-PAGE上樣緩沖液變性。 1.3 M1受體、B2受體及相關(guān)信號(hào)分子在膜脂筏中分布的鑒定 將超速離心后的九部分及去污劑溶解部分與不溶部分經(jīng)12%的SDS-聚丙烯酰胺凝膠電泳分離后電轉(zhuǎn)移至NC膜。膜用5%的脫脂奶粉常溫下封閉1小時(shí)。分別加特異性的抗體孵育過(guò)夜�？贵w有單克隆抗體caveolin-1(1:1000), B2受體(1:1000),多克隆抗體M1(1:200),Gq(1:200),G11(1:200),PLCβ4(1:200),PLCβ1(1:200)。然后用TBST洗膜液洗膜三次,每次10分鐘。加相應(yīng)的熒光二抗(1:4000),室溫孵育3-4小時(shí)。洗膜后用Odyssey9120雙色紅外激光成像系統(tǒng)顯色分析。 2 M電流記錄 分離培養(yǎng)頸上神經(jīng)節(jié)神經(jīng)元,打孔膜片鉗記錄M電流。將細(xì)胞鉗制在-20 mV,然后復(fù)極化到-60 mV,記錄膜電位在-60 mV時(shí)M電流的尾電流。比較甲基環(huán)糊精(MβCD)處理前后給予OXO-M (5μM)和BK(100 nM)后M電流的抑制情況。 3細(xì)胞內(nèi)Ca~(2+)測(cè)定 使用熒光探針Flu-4-AM(2.5 nmol/L)和透膜劑F127(0.02%)標(biāo)記鈣離子,室溫孵育30分鐘,使染料進(jìn)入細(xì)胞。用0.01 M PBS清洗細(xì)胞三次,然后用激光共聚焦顯微鏡觀察環(huán)糊精處理前后再給予緩激肽刺激時(shí)的細(xì)胞內(nèi)鈣離子的變化情況。 4免疫細(xì)胞化學(xué)和免疫共沉淀 4.1免疫細(xì)胞化學(xué)觀察caveolin-1與B_2受體和M1受體的共定位。將細(xì)胞用4%多聚甲醛固定后,用含0.2% Triton-100的PBS作用30分鐘,增加通透性,然后加入特異性的一抗和熒光標(biāo)記的二抗或三抗,用激光共聚焦掃描顯微鏡觀察鼠cavolin-1與鼠B2受體和兔M1受體的共定位情況。 4.2免疫共沉淀方法觀察頸上神經(jīng)節(jié)中IP3受體與B2受體之間的相互關(guān)系。摘取SCG組織并用玻璃勻漿器勻漿后,離心5000 rpm,10 min。取上清,加入2μl IP3抗體,4℃,60 rpm振搖過(guò)夜,次日加入protein G beads, 4℃,60 rpm,4 h后洗滌珠子,將純化后的蛋白免疫復(fù)合物進(jìn)行SDS-PAGE凝膠電泳,電轉(zhuǎn)移至NC膜上,用脫脂奶粉封閉后,用B2抗體和熒光染料標(biāo)記的二抗進(jìn)行檢測(cè),顯色結(jié)果用Odyssey9120雙色紅外激光成像系統(tǒng)掃描分析。 結(jié)果: 1在大鼠頸上神經(jīng)節(jié)中有脂筏的存在,其特異性蛋白caveolin-1分布于蔗糖密度梯度離心分離組份的第3,4,5層(共9層),且第4層含量最多。脂筏的另一標(biāo)記物GM1分布于1-5層,其中在1-3層含量較多。 2 B_2受體、Gq、G_(11)、PLCβ_4和PLCβ_1都在脂筏中有分布,而M1受體少見于脂筏中。 3用1% triton X-100處理膜樣品之后,在不溶解于去污劑的部分中通過(guò)Western blots可以檢測(cè)到caveolin-1、B2受體、M1受體、PLCβ1、PLCβ4、Gq、G11的存在。就B2受體和M1受體而言, B2受體在去污劑不溶的部分中的比例高達(dá)83.9%,遠(yuǎn)高于M1受體的14.7%。其它在去污劑不溶的部分中的比例為:PLCβ4 19.7%,PLCβ1 18.6%,Gq 34%,G11 21.6%。在用5 mM MβCD處理1h后,B2受體在去污劑不溶的部分的含量降至原來(lái)的30%(P0.05,n=3)。 4 M受體激動(dòng)劑OXO-M誘導(dǎo)的SCG神經(jīng)元M電流抑制的百分率在給予MβCD前為82±6.3%,而在給予10 mM MβCD和5 mM MβCD分別為49±8.7%(與給MβCD前相比顯著減少,P0.01)和71±4.9%(與給MβCD前相比無(wú)顯著差別,P0.05);而緩激肽BK誘導(dǎo)的M電流抑制的百分率在給予MβCD前為77±7%,而在給予MβCD 5 mM和10 mM MβCD后分別為32±8.5%(與給MβCD前相比顯著減少,P 0.01)和37±8%(與給MβCD前相比顯著減少, P 0.01),表明BK誘導(dǎo)的M電流抑制對(duì)MβCD更敏感。 5 5 mM MβCD處理SCG神經(jīng)元后明顯減弱BK誘導(dǎo)的細(xì)胞內(nèi)鈣升高,給予MβCD前后BK誘發(fā)的細(xì)胞內(nèi)鈣的變化率分別為128.3±3.1%和107.4±3.0%(P 0.01)。 6在SCG神經(jīng)元B_2受體較之M1受體與caveolin-1有更多共定位現(xiàn)象。 結(jié)論: 1大鼠頸上神經(jīng)節(jié)(SCG)組織細(xì)胞上存在脂筏微域,Ⅱ型緩激肽受體(B_2)及其相關(guān)細(xì)胞信號(hào)通路分子如Gq、G11、PLCβ4和PLCβ1都在脂筏中有分布;M1受體可能不存在于脂筏中 2脂筏在B_2受體調(diào)節(jié)SCG神經(jīng)元M通道功能中發(fā)揮重要作用 3緩激肽受體(B_2)激活后升高SCG神經(jīng)元內(nèi)鈣需要脂筏的空間完整性 4緩激肽受體(B_2)與caveolin-1在SCG神經(jīng)元有共定位
[Abstract]:M current is a potassium current widely exist in the nervous system, is one of the main mechanisms of neuronal excitability, is only activated in neurons near the threshold potential of current, it can cause the function to reduce neuronal hyperexcitability, epilepsy and other diseases. Many neurotransmitters and neuropeptides can affect the excitability of M current regulation neurons. In recent years, great progress has the molecular mechanisms that regulate the M current of neurotransmitters, including acetylcholine activated type M cholinergic receptor (M1) and bradykinin activates its II bradykinin receptor (B2 receptor) molecular mechanism regulating M current is the most representative. Although the activation of two receptors can inhibit M current, but the molecular mechanism is different, the effects on intracellular Ca2+ concentration is different key points. However, for the different further mechanism will require further research. The experiment attempts to explain the different mechanism of the cell signaling pathway mediated by different membrane receptors on the current regulation of M from the angle of cell membrane lipid rafts (lipid raft).
Lipid raft is a lipid membrane microdomains that are distributed in the cell membrane of most mammals, the size is 55-300 nm, rich in cholesterol and sphingomyelin, has a low buoyant density and insoluble in detergent properties. Recent studies have shown that lipid rafts play an important role in signal transduction events in different signal; specific molecular aggregation in lipid rafts may determine the specificity of different signal transduction pathways. In this study with the M1 receptor, B2 receptor and the differences of the distribution related signaling molecules in the lipid rafts as the center, to observe the role of lipid rafts in the regulation of specific M in the current.
Objective: To study the role of lipid rafts in the rat superior cervical ganglion cell membrane in the specific regulation of M current by membrane receptor
Method:
Identification of membrane proteins in 1 lipid rafts and lipid rafts
Isolation of lipid rafts and lipid rafts by 1.1 sucrose density gradient centrifugation
The removal of 40 SD rats of 7 days SCG, were homogenized with a glass homogenizer and centrifuged for 5000 rpm, 5 min. of the supernatant. About 1.5 ml in the supernatant of 8.9 ml centrifuge tube, adding 80% sucrose 1.5 ml, mix gently to avoid bubbles, above the surface and slowly add 35% sucrose 3 ml, with the same method the operation, adding about 3 ml 5%.39000 RPM sucrose, sucrose gradient, the preparation of 40%/35%/5% 4 C, 21 h. After the centrifugal centrifugal liquid from top to bottom out continuously in nine parts, each part is about one out of 4 L ml. In each part, the uniform location on the nylon membrane, Dot blots experiment, determination of ganglioside GM1 in the distribution of the nine parts of the cholera toxin B, and GM1 is one of the molecular markers of lipid rafts.
Separation of lipid rafts by 1.2 detergents
The removal of 10 SD rats of 7 days SCG, with a glass homogenizer homogenate, centrifuged for 5000 rpm, 10 min, the supernatant, 40200 rpm, 4 C, 1 h. precipitation with 200 L containing 500 mM Na2CO3 and 1% Triton low permeability homogenate Buffer suspension and ultrasound on ice, standing for 30 min, 15000 G. 4 C, 30 min, decontamination agent into soluble and insoluble two components, and then use the 5 * SDS-PAGE sample buffer degeneration.
Identification of the distribution of 1.3 M1 receptor, B2 receptor and related signal molecules in membrane lipid rafts
The nine part and the detergent solubilized part after ultracentrifugation and insoluble fraction by 12% SDS- polyacrylamide gel electrophoresis after electro transferred to NC membrane. The membrane with 5% skim milk at room temperature for 1 hours respectively. The antibody was closed to specific fertility overnight. Antibody monoclonal antibody caveolin-1 (1:1000), B2 receptor (1:1000), M1 polyclonal antibody (1:200), Gq (1:200), G11 (1:200), PLC (1:200) PLC beta 4, beta 1 (1:200). Then washed with TBST film three times liquid membrane cleaning, every 10 minutes. Add the corresponding fluorescence resistance (1:4000), two 3-4 after incubation at room temperature hour. Wash the membrane with Odyssey9120 dual color infrared laser imaging system color analysis.
2 M current record
Cultured superior cervical ganglion neurons, perforated patch clamp recording M current. The cells held at -20 mV, -60 mV and repolarization, the tail current recording of membrane potential in -60 mV M current. Comparison of methyl cyclodextrin (M beta CD) before and after treatment with OXO-M (5 M) and BK (100 nM) the inhibition of M currents.
Determination of Ca~ (2+) in 3 cells
The use of fluorescence probe Flu-4-AM (2.5 nmol/L) and penetrating agent F127 (0.02%) labeled calcium ions, incubated at room temperature for 30 minutes, the dye into the cell. Three M 0.01 PBS cells were washed, then the changes of microscopic observation of cyclodextrin before and after treatment to give bradykinin stimulation in the intracellular calcium confocal laser.
4 immunocytochemistry and immunoprecipitation
4.1 immunocytochemical co localization of caveolin-1 and B_2 receptor and M1 receptor. The cells were fixed with 4% paraformaldehyde, with PBS containing 0.2% Triton-100 30 minutes, increase the permeability, then add specific antibody and FITC labeled anti two or three resistance, the co localization observation of mouse cavolin-1 and mouse B2 receptor and M1 receptor in rabbit by confocal laser scanning microscope.
4.2 co immunoprecipitation method between cervical ganglia IP3 and B2 receptors. The relationship between SCG and the removal of tissue homogenate after centrifugal glass homogenizer, 5000 rpm, 10 min. supernatant, adding 2 L IP3 antibody, 4 C, 60 rpm shaking overnight, the next day to join protein G beads, 4 C 60, RPM, 4 h after washing the beads, the immune complex protein purified by SDS-PAGE gel electrophoresis and electrotransferred to NC membrane, the skimmed milk is closed, with B2 antibody and fluorescent dye labeled two antibody detection, color results with two-color infrared laser imaging system Odyssey9120 scanning analysis.
Result:
1, there were lipid rafts in the superior cervical ganglion of rats. The specific protein caveolin-1 was distributed in the 3,4,5 layer (9 layers) of sucrose density gradient centrifugation, and the fourth layer was the most. The other marker GM1 of lipid rafts was distributed on the 1-5 level, which contained more in the 1-3 level.
2 B_2 receptors, Gq, G_ (11), PLC beta _4 and PLC beta _1 are all distributed in lipid rafts, and M1 receptors are rarely found in lipid rafts.
3 with 1% Triton X-100 film samples, without detergent dissolved in part by Western blots can detect caveolin-1, B2 receptor, M1 receptor, PLC PLC beta 1, beta 4, Gq, G11. B2 and M1 receptor, B2 receptor in detergent insoluble part in the proportion as high as 83.9%, much higher than that of M1 receptor 14.7%. other insoluble in detergent section ratio: PLC PLC beta 419.7%, beta 118.6%, Gq 34%, G11 21.6%. in mM M 5 beta CD after 1h treatment, B2 receptor in detergent insoluble part of the content to the original 30% (P0.05, n=3).
4 the percentage of SCG neurons with M receptor agonist M current agent OXO-M induced inhibition of M in beta CD for 82 + 6.3%, and received 10 mM and 5 mM M beta CD M beta CD were 49 + 8.7% (compared to M beta CD significantly decreased, P0.01 and 71 + 4.9% (compared) and to M beta before CD had no significant difference, P0.05 and M); the percentage of current BK bradykinin induced inhibition of M in beta CD for 77 + 7%, while in the given M beta CD 5 mM and 10 mM respectively after CD M beta 32 + 8.5% (compared to M beta CD significantly decreased P, 0.01) and 37 + 8% (compared to M beta CD significantly decreased, P 0.01), M current showed that BK induced inhibition was more sensitive to M beta CD.
After 55 mM M beta CD treatment of SCG neurons, the intracellular calcium increased significantly after BK treatment, and the changes of intracellular calcium induced by BK before and after M CD CD were 128.3 + 3.1% and 107.4 + 3% (P 0.01), respectively.
6 more co localization of B_2 receptor in SCG neurons than that of M1 receptor and caveolin-1.
Conclusion:
There are lipid rafts in the 1 cervical ganglion neurons (SCG). The B_2 receptor and its related cellular signaling pathways, such as Gq, G11, PLC beta 4 and PLC beta 1, are distributed in lipid rafts. M1 receptors may not exist in lipid rafts.
2 lipid rafts play an important role in the function of B_2 receptor to regulate the function of M channel in SCG neurons
3 bradykinin receptor (B_2) activation increases the spatial integrity of calcium in SCG neurons requiring lipid rafts
4 bradykinin receptor (B_2) and caveolin-1 have co localization in SCG neurons

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R331

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8 吳文杰;陽(yáng)喬;曹芹芳;張耀文;唐望先;;FAAH對(duì)內(nèi)源性大麻素作用肝細(xì)胞的保護(hù)作用機(jī)制研究[A];第十八次全國(guó)中西醫(yī)結(jié)合肝病學(xué)術(shù)會(huì)議論文匯編[C];2009年

9 鐘建;沙印林;;脂筏模型膜系統(tǒng)的建立[A];中國(guó)科協(xié)2005年學(xué)術(shù)年會(huì)生物物理與重大疾病分會(huì)論文摘要集[C];2005年

10 何會(huì);葉紅;金肆;馬萬(wàn)里;呂麗華;劉聲遠(yuǎn);王迪潯;胡清華;;脂筏聚集及其與CaSR,FKBP12.6的共定位在哮喘細(xì)胞因子所致氣道上皮細(xì)胞鈣振蕩中的作用[A];湖北省暨武漢市病理生理學(xué)會(huì)第十四屆學(xué)術(shù)年會(huì)論文匯編[C];2008年

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1 張婷;基于生物分子網(wǎng)絡(luò)的細(xì)胞脂筏和TRAIL信號(hào)通路的研究[D];清華大學(xué);2011年

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3 黃紅蘭;脂筏在人類皰疹病毒6型裝配中的作用及對(duì)病毒感染的影響[D];吉林大學(xué);2006年

4 朱勇U，

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