脂筏在细胞信号特异性调节M通道功能中的作用

发布时间：2018-01-19 20:23

本文关键词： 脂筏颈上神经节 M_1受体 B_2受体 M电流信号分子甲基-β-环糊精　出处：《河北医科大学》2011年硕士论文　论文类型：学位论文

【摘要】：M电流是广泛存在于神经系统的一种钾电流,是影响神经元兴奋性的主要机制之一,是唯一在神经元阈电位附近激活的电流,它的功能降低可以引起神经元兴奋性增高,诱发癫痫等疾病。众多神经递质和神经肽可以调节M电流进而影响神经元兴奋性。近年来,对神经递质等调节M电流的分子机制有了很大进展,其中以乙酰胆碱激活M型胆碱受体(M1)和缓激肽激活其II型缓激肽受体(B2受体)后调节M电流的分子机制最有代表性。虽然两种受体激活都可以抑制M电流,但其分子机制有所不同,其中对细胞内Ca2+浓度影响的不同是关键之处。然而,对于造成这一不同的进一步机制则有待进一步研究。本实验试图从细胞膜脂筏(lipid raft)的角度来解释不同膜受体介导的细胞信号通路对M电流调节不同的机制。脂筏是细胞膜上的一种脂质微区,在大多数哺乳动物的细胞膜上都有分布,一般大小为55-300 nm,富含胆固醇和鞘磷脂,具有低浮力密度和不溶于去污剂的特性。近期研究表明,脂筏在信号转导事件中发挥着重要的作用;脂筏中特异聚集的不同信号分子可能决定了不同信号转导通路的特异性。本研究将以与M1受体、B2受体以及所相关的细胞信号分子在脂筏中分布的异同为中心,观察脂筏在M电流的特异性调节中的作用。目的:研究大鼠颈上神经节神经元细胞膜脂筏在膜受体特异调节M电流中的作用方法: 1脂筏提取及脂筏中膜蛋白的鉴定 1.1蔗糖密度梯度离心法分离脂筏及脂筏的鉴定摘取40只7天SD大鼠SCG,用玻璃匀浆器进行匀浆,离心5000 rpm,5 min。取上清。将约1.5 ml上清置于8.9 ml离心管底部,加入80%蔗糖1.5 ml,轻轻混匀避免气泡的产生,在液面上方缓慢加入3 ml 35%蔗糖,同法操作,再加入约3 ml 5%蔗糖,制备40%/35%/5%的蔗糖梯度。39,000 rpm,4℃,离心21 h。将离心后的液体从上到下连续取出九个部分,每部分约一毫升。从每个部分中取出4μl,均匀地点于尼龙膜上,进行Dot blots实验,利用霍乱毒素B测定神经节苷脂GM1在九个部分的分布情况,而GM1是脂筏的标记分子之一。 1.2去污剂法分离脂筏摘取10只7天SD大鼠SCG,用玻璃匀浆器匀浆,离心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低渗匀浆Buffer悬浮并超声,冰上静置30 min,15,000 g, 4℃,30 min,分成去污剂溶解与不溶两种组分,然后用5×SDS-PAGE上样缓冲液变性。 1.3 M1受体、B2受体及相关信号分子在膜脂筏中分布的鉴定将超速离心后的九部分及去污剂溶解部分与不溶部分经12%的SDS-聚丙烯酰胺凝胶电泳分离后电转移至NC膜。膜用5%的脱脂奶粉常温下封闭1小时。分别加特异性的抗体孵育过夜。抗体有单克隆抗体caveolin-1(1:1000), B2受体(1:1000),多克隆抗体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分钟。加相应的荧光二抗(1:4000),室温孵育3-4小时。洗膜后用Odyssey9120双色红外激光成像系统显色分析。 2 M电流记录分离培养颈上神经节神经元,打孔膜片钳记录M电流。将细胞钳制在-20 mV,然后复极化到-60 mV,记录膜电位在-60 mV时M电流的尾电流。比较甲基环糊精(MβCD)处理前后给予OXO-M (5μM)和BK(100 nM)后M电流的抑制情况。 3细胞内Ca~(2+)测定使用荧光探针Flu-4-AM(2.5 nmol/L)和透膜剂F127(0.02%)标记钙离子,室温孵育30分钟,使染料进入细胞。用0.01 M PBS清洗细胞三次,然后用激光共聚焦显微镜观察环糊精处理前后再给予缓激肽刺激时的细胞内钙离子的变化情况。 4免疫细胞化学和免疫共沉淀 4.1免疫细胞化学观察caveolin-1与B_2受体和M1受体的共定位。将细胞用4%多聚甲醛固定后,用含0.2% Triton-100的PBS作用30分钟,增加通透性,然后加入特异性的一抗和荧光标记的二抗或三抗,用激光共聚焦扫描显微镜观察鼠cavolin-1与鼠B2受体和兔M1受体的共定位情况。 4.2免疫共沉淀方法观察颈上神经节中IP3受体与B2受体之间的相互关系。摘取SCG组织并用玻璃匀浆器匀浆后,离心5000 rpm,10 min。取上清,加入2μl IP3抗体,4℃,60 rpm振摇过夜,次日加入protein G beads, 4℃,60 rpm,4 h后洗涤珠子,将纯化后的蛋白免疫复合物进行SDS-PAGE凝胶电泳,电转移至NC膜上,用脱脂奶粉封闭后,用B2抗体和荧光染料标记的二抗进行检测,显色结果用Odyssey9120双色红外激光成像系统扫描分析。结果: 1在大鼠颈上神经节中有脂筏的存在,其特异性蛋白caveolin-1分布于蔗糖密度梯度离心分离组份的第3,4,5层(共9层),且第4层含量最多。脂筏的另一标记物GM1分布于1-5层,其中在1-3层含量较多。 2 B_2受体、Gq、G_(11)、PLCβ_4和PLCβ_1都在脂筏中有分布,而M1受体少见于脂筏中。 3用1% triton X-100处理膜样品之后,在不溶解于去污剂的部分中通过Western blots可以检测到caveolin-1、B2受体、M1受体、PLCβ1、PLCβ4、Gq、G11的存在。就B2受体和M1受体而言, B2受体在去污剂不溶的部分中的比例高达83.9%,远高于M1受体的14.7%。其它在去污剂不溶的部分中的比例为:PLCβ4 19.7%,PLCβ1 18.6%,Gq 34%,G11 21.6%。在用5 mM MβCD处理1h后,B2受体在去污剂不溶的部分的含量降至原来的30%(P0.05,n=3)。 4 M受体激动剂OXO-M诱导的SCG神经元M电流抑制的百分率在给予MβCD前为82±6.3%,而在给予10 mM MβCD和5 mM MβCD分别为49±8.7%(与给MβCD前相比显著减少,P0.01)和71±4.9%(与给MβCD前相比无显著差别,P0.05);而缓激肽BK诱导的M电流抑制的百分率在给予MβCD前为77±7%,而在给予MβCD 5 mM和10 mM MβCD后分别为32±8.5%(与给MβCD前相比显著减少,P 0.01)和37±8%(与给MβCD前相比显著减少, P 0.01),表明BK诱导的M电流抑制对MβCD更敏感。 5 5 mM MβCD处理SCG神经元后明显减弱BK诱导的细胞内钙升高,给予MβCD前后BK诱发的细胞内钙的变化率分别为128.3±3.1%和107.4±3.0%(P 0.01)。 6在SCG神经元B_2受体较之M1受体与caveolin-1有更多共定位现象。结论: 1大鼠颈上神经节(SCG)组织细胞上存在脂筏微域,Ⅱ型缓激肽受体(B_2)及其相关细胞信号通路分子如Gq、G11、PLCβ4和PLCβ1都在脂筏中有分布;M1受体可能不存在于脂筏中 2脂筏在B_2受体调节SCG神经元M通道功能中发挥重要作用 3缓激肽受体(B_2)激活后升高SCG神经元内钙需要脂筏的空间完整性 4缓激肽受体(B_2)与caveolin-1在SCG神经元有共定位
[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

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R331

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