【作者】 崔艺峰；

【导师】 许文燮； 李在琉；

【作者基本信息】 延边大学 ， 消化生理， 2003， 硕士

【摘要】 不饱和脂肪酸在牵张增强豚鼠胃窦平滑肌细胞毒蕈碱电流中的作用生物膜是一个复杂的多分子体系，1972年，Nicolson提出“液态镶嵌学说”以来，这个理论从多方面得到了证实，其基本观点是：膜蛋白镶嵌在膜脂中，膜蛋白与膜脂具有相互作用。作为膜脂重要成分的脂肪酸对膜蛋白结构与功能的影响是非常重要的。长链多不饱和脂肪酸主要包括n-3类和n-6类，其中最具代表性的是花生四烯酸（arachidonic acid，AA）、亚麻酸（linoleic acid，LA）、亚油酸（oleic acid，OA）。许多研究表明这些不饱和脂肪酸对离子通道既有激活作用又有抑制作用，其作用非常复杂。它们对广泛存在于各种细胞膜表面的离子通道进行直接或间接的调控作用，如花生四烯酸与离子通道的直接作用改变了通道蛋白的构象；花生四烯酸还可以通过代谢产物、蛋白激酶C（PKC）和 Ca2+间接影响离子通道，从而参与调节胃肠平滑肌的肌源性收缩与舒张。最近，机械牵张可调节多种离子通道功能的事实已被很多学者所证实，机械牵张可以激活细胞内的众多信号传导途径。但是，要明确究竟哪种途径在参<WP=7>与机械信号的感知和在细胞内的信号传递过程中起决定性的作用，较为困难。关于细胞膜如何感知机械牵拉和将机械牵拉转变为生物化学信号，机制较为复杂。机械牵拉刺激可能通过整合素（integrin），细胞骨架（cytoskeleton）或者一些特殊蛋白，如磷脂酶、离子通道、蛋白激酶等与细胞内信号传导途径偶联在一起，最终导致细胞膜各种离子通道功能的改变。但是也有可能在没有细胞内信号传导途径参与下通过细胞骨架直接改变离子通道的功能。 本实验在急性分离的豚鼠胃窦环行肌细胞上， 利用膜片钳技术的全细胞记录法观察了外源性不饱和脂肪酸对豚鼠胃窦平滑肌细胞毒蕈碱电流的影响，及其作用机制， 并且探讨了外源性不饱和脂肪酸是否参与低渗牵张加强毒蕈碱电流的过程。本文的主要实验结果如下：1. 不饱和脂肪酸对毒蕈碱电流的影响1.1 膜电位钳制在-20.0mV，用Cs+代替细胞内外的K+和Na+阻断K+通道时，50?mol?L-1 卡巴胆碱（Carbachol，CCh）可诱导内向性毒蕈碱电流(ICCh)， 其平均幅度为183.3±30.7pA。花生四烯酸（arachidonic acid，AA）明显抑制ICCh，并具有量效关系。当AA的浓度在1、3和5 ?mol?L-1时，分别抑制ICCh至46 %±8 %、23 %±5 % 和 3.8 %±0.9 %。在同样实验条件下， 另一种不饱和脂肪酸，亚麻酸（linoleic acid，LA）也抑制ICCh，而且也有明显量效关系。在 1、5和10 ?mol?L-1浓度时<WP=8>1.2 分别抑制ICCh至69 %±10 %、35 %±5 % 和 7.4 % ±1.2 %。1.3 为探讨脂肪酸对ICCh抑制效应与不饱和脂肪酸的不饱和度之间的关系， 在本实验中观察了5 μmol?L-1的AA、LA和亚油酸（oleic acid，OA）对ICCh的影响。 结果发现，它们分别抑制ICCh至3.8 %±0.9 %、35 %±5 %和67 % ±9 %，即AA（C20: 4, cis-5, 8, 11, 14）>LA(C18: 2, cis-9, 12)>OA (C18: 1, cis-9)。1.4 不饱和脂肪酸抑制ICCh，那么不饱和脂肪酸对ICCh的作用是通过直接途径还是通过其他间接途径来实现的？为了阐明不饱和脂肪酸对ICCh的作用机制，在本实验中用H-7（蛋白激酶C抑制剂）100 ?mol?L-1或消炎痛（环氧化酶抑制剂）10 ?mol?L-1预处理10 -15 分以后， 观察了AA对ICCh的作用。AA分别抑制ICCh至5.5 %±0.7 % 和3.0 %±1.0%。这个结果与对照值没有显著性的差异。2. 不饱和脂肪酸在低渗加强毒蕈碱电流中的作用2.1 当膜电位钳制在-20.0mV时，50?mol?L-1卡巴胆碱（carbachol，CCh）能够诱导毒蕈碱电流（ICCh）。 低渗牵张可以使ICCh明显增加到对照的226.0%±21.0%。为探讨花生四烯酸等不饱和脂肪酸是否通过磷脂酶途径参与调节低渗增加ICCh过程？本实验先用5?mol?L-1花生四烯酸（arachidonic acid，<WP=9>2.2 AA）、亚麻酸（linoleic acid，LA）和亚油酸（oleic acid，OA）预处理以后观察了低渗对ICCh的作用。结果表明，ICCh 先分别被AA、LA和OA抑制到对照的3.8%±0.6%、 35.2%±0.8%和66.6%±0.6%。在分别用AA、LA和OA（5?mol?L-1）预处理情况下，再给予低渗刺激可以使ICCh分别增加到 106.0%±2.5%、173.2%±6.8% 和 222.1%±11.0%。随着不饱和脂肪酸的不饱和度不同，低渗加强ICCh的程度也不同。以上结果可能暗示，不饱和脂肪酸只对ICCh本身起作用，但可能不参与调节低渗增强ICCh过程。2.3 为了进一步证实不饱和脂肪酸参与低渗加强毒蕈碱电流的与否，先用低渗牵张加强毒蕈碱电流之后，再用含有5?mol?L-1 AA的低渗液灌流时，AA只抑制了51.2%±3.8%，而同一浓度AA在等渗状态下抑制了ICCh 96.2%±1.6%。上述结果提示：1）不饱和脂肪酸抑制毒蕈碱电流，且有量效关系；2）不饱和脂肪酸中双键数目越多，抑制效应越强；3）不饱和脂肪酸抑制ICCh不通过其代谢产物途径和细胞内信号传导系统等间接途径，可能是直接作用于离子通道；4）不饱和脂肪酸不参与低渗刺激加强毒蕈碱电流过程。更多还原

【Abstract】 Role of unsaturated fatty acid in stretch-enhanced muscarinic current in guinea-pig gastric myocytesPlasma membrane is a complex excessive molecule style. Since Nicolson mentioned "Fluid mosaic theory" in 1972 and it was confirmed from many aspect, its basic viewpoint is that membrane protein mounts in the membrane lipid, and they have mutual action. As a ingredient of membrane lipid fatty acids have effect on membrane protein and modulate its construction and function. Unsaturated fatty acids include type n-3 and type n-6, among of them the representative fatty acids are arachidonic acid (AA), linoleic acid (LA) and oleic acid (OA) so on. Many investigation demonstrated that unsaturated fatty acids either active or inhibit ion channels. The effect of fatty acids on ion channel are complicated: fatty acid regulation of ion channels is either direct or indirect. The direct modulation changes the conformation of ion channel protein by the interaction between fatty acid and the protein, or through the interference with cell membranes around channels. The indirect influence of arachidonic acid upon channels results from its metabolites caused by cylccoxygenase and lipooxygenase, protein kinase C (PKC)<WP=11>and intracellular calcium. More recently, mechanical stretch could regulate numerous ion channels function had been confirmationed by many scholar. The mechanical stretch could active numerous intracellular signal pathway, but it is quite difficult to make definite, on earth which signal transduction pathways may couple mechanical stress. The mechanism is more complicated how mechanical stress may be sensed and changed it to biochemistry signal. Mechanical stress may be coupled to intracellular signals via integrins and the cytoskeleton or via sarcolemmal proteins, such as phospholipases, ion channels and ion exchangers, finally, increase intracellular Ca2+ concentration. But stress signal may be directly change the ion channel function via the cytoskeleton without the involvement of signal transduction pathways.In this paper we investigated the effect of AA on muscarinic current and its mechanism, also observed whether exogenous unsaturated fatty acid participated in the hyposmotic membrane stretch induced-increase of muscarinic current (ICCh) in gastric antral circular myocytes of guinea-pig.1. Effect of unsaturated fatty acid on muscarinc current(ICCh)Under the whole-cell configuration, the membrane potential was clamped at -20.0mV, and sodium chloride in external solution and potassium chloride in<WP=12>1.1 the pipette solution were replaced by CsCl to block potassium channel, and ICCh was elicited by 50μmol?L-1 Carbachol. AA, an unsaturated fatty acid (with four double bonds) significantly inhibited ICCh in a dose-dependant manner. AA respectively inhibited ICCh to 46%±8%, 23%±5% and 3.8%±0.9% at concentration of 1, 3 and 5 μmol?L-1.1.2 And under the same condition, another unsaturated fatty acids, linoleic acid(LA) also inhibited ICCh in a dose-dependant manner, LA respectively inhibited to 69.17%±0.45%, 35.2%±0.79% and 7.41%±0.15% at 1, 5 and 10?mol?L-1.1.3 Under the whole-cell configuration, same concentration (5μmol?L-1) of AA, LA and OA (with one double bond) inhibited ICCh to 3.76%±0.64%, 35.2±0.79% and 66.62%±0.61% respectively. Compared both of them mutually, the inhibitory potency sequence was AA (C20: 4, cis-5, 8, 11, 14)>LA (C18: 2, cis-9, 12)>OA (C18: 1, cis-9).To determine whether the inhibitory effects of unsaturated fatty acids on ICCh is direct or indirect, the effect of AA on ICCh was observed after pretreated with indomethacin and H-7 aboout10-15 minutes. In the presence of 100?mol?L-1 H-7(protein phosphorylation C inhibitor) and 10?mol?L-1 indomethacin (cyclooxygenase inhibitor), AA still inhibited ICCh to 5.5%±<WP=13>1.4 0.2% and 3.04%±0.5% respectively. Each of them compared with the effect of AA on ICCh, there were no significant difference in the inhibitory effect of AA on ICCh before pretreatment with H-7 and indomethacin and that o更多还原