酪氨酸家族激酶在大鼠脊髓背角LTP中的作用及其機(jī)制

鐘 祎， 黃陽亮， 許繼德

(1廣州醫(yī)學(xué)院生理教研室，廣東 廣州 510182 ；2中山大學(xué)附屬第一醫(yī)院脊柱外科，廣東 廣州 510700)

酪氨酸家族激酶在大鼠脊髓背角LTP中的作用及其機(jī)制

鐘 祎1△， 黃陽亮2， 許繼德1

(1廣州醫(yī)學(xué)院生理教研室，廣東 廣州 510182 ；2中山大學(xué)附屬第一醫(yī)院脊柱外科，廣東 廣州 510700)

目的探討酪氨酸家族激酶(SFKs)在大鼠脊髓背角C纖維誘發(fā)電位的長時(shí)程增強(qiáng)(LTP)中的作用及其機(jī)制。方法用在體電生理方法檢測SFKs抑制劑對高頻刺激坐骨神經(jīng)誘導(dǎo)的脊髓背角LTP的影響;用Western blotting方法檢測高頻刺激誘導(dǎo)脊髓LTP的不同時(shí)點(diǎn)大鼠脊髓背角磷酸化SFKs的表達(dá)情況;用免疫熒光雙染方法檢測高頻刺激誘導(dǎo)脊髓LTP后大鼠脊髓背角磷酸化SFKs表達(dá)的細(xì)胞學(xué)定位。結(jié)果SFKs的抑制劑(PP2或SU6656)可以完全阻斷LTP的誘導(dǎo),甚至將LTP逆轉(zhuǎn)為長時(shí)程抑制(LTD);高頻電刺激大鼠坐骨神經(jīng)誘導(dǎo)脊髓LTP后15 min開始,刺激同側(cè)的脊髓背角中磷酸化SFKs表達(dá)明顯增加,并且這些高表達(dá)的磷酸化SFKs僅僅存在于脊髓小膠質(zhì)細(xì)胞中。結(jié)論在脊髓背角,小膠質(zhì)細(xì)胞中的SFKs 是誘導(dǎo)LTP的必要條件,抑制SFKs及其下游分子可能有助于治療病理性疼痛。

Src家族激酶類; 長時(shí)程增強(qiáng); 疼痛; 脊髓背角

C纖維誘發(fā)電位的長時(shí)程增強(qiáng)(long-term potentiation, LTP)是指傳入C纖維與脊髓背角神經(jīng)元之間突觸傳遞效率的持續(xù)增強(qiáng),以前的研究發(fā)現(xiàn)電刺激傳入C纖維以及末梢神經(jīng)損傷可以誘導(dǎo)脊髓背角LTP[1, 2]。由于傳入C纖維傳遞痛覺信息并與脊髓淺層神經(jīng)元形成突觸聯(lián)系,而且誘導(dǎo)LTP的刺激可以在人身上引起長時(shí)間的痛覺過敏和痛覺超敏[3],脊髓LTP被認(rèn)為是一種痛覺記憶,是目前研究病理性疼痛的突觸模型[4,5]。酪氨酸家族激酶(Src-family kinases, SFKs)廣泛表達(dá)于哺乳動(dòng)物的中樞神經(jīng)系統(tǒng),參與了神經(jīng)系統(tǒng)的增生和分化[6,7],最近的研究表明末梢神經(jīng)損傷激活脊髓中的SFKs, 并且活化的SFKs僅僅存在于脊髓小膠質(zhì)細(xì)胞中;鞘內(nèi)給予SFKs的抑制劑PP2可以阻斷神經(jīng)損傷引起的痛覺過敏的產(chǎn)生,這說明SFKs在病理性疼痛的產(chǎn)生中起重要作用。本研究觀察SFKs抑制劑對脊髓LTP誘導(dǎo)的影響,以及脊髓LTP誘導(dǎo)后脊髓背角SFKs磷酸化水平的變化。

材 料 和 方 法

1材料

1.1動(dòng)物 實(shí)驗(yàn)采用Sprague-Dawley雄性大鼠(250-280 g), 由廣州醫(yī)學(xué)院實(shí)驗(yàn)動(dòng)物中心提供。動(dòng)物分籠飼養(yǎng),自由飲食,室溫保持在(24±1) ℃, 濕度50%-60%。

1.2主要試劑 PP2、SU6656 和PP3 (Calbiochem)首先溶解在二甲基亞砜(dimethyl sulphoxide, DMSO)中配成50 mmol/L的儲存液,分裝儲存在-80 ℃冰箱中,使用前用生理鹽水稀釋成所需濃度。工作液中DMSO的濃度低于0.5% (V/V)。多克隆兔抗大鼠 p-SFKs抗體、多克隆兔抗大鼠 SFKs 抗體和多克隆兔抗大鼠 β-actin 抗體均購自Cell Signalling Technology; 抗-神經(jīng)元特異性核蛋白抗體(neuronal-specific nuclear protein, NeurN)、抗-膠質(zhì)細(xì)胞纖維酸性蛋白抗體(glial fibrillary acidic protein, GFAP)和抗-小膠質(zhì)細(xì)胞抗體OX-42均購自Chemicon。辣根過氧化酶(HRP) 標(biāo)記的山羊抗兔IgG購自Santa Cruz; FITC 標(biāo)記的Ⅱ抗和CY3 標(biāo)記的Ⅱ抗購自Jackson Immunolab。

2方法

2.1在體電生理記錄 烏拉坦(1.5 g/kg)腹腔注射麻醉，行氣管插管使大鼠自然呼吸；一側(cè)頸動(dòng)脈插管持續(xù)監(jiān)測大鼠血壓(波動(dòng)于80 mmHg-120 mmHg)；在腰4-腰6(L4-L6)間行椎板切除術(shù)，暴露腰膨大。游離左側(cè)坐骨神經(jīng)，使用雙極氯化銀電極刺激坐骨神經(jīng)。除用于記錄的脊髓節(jié)段外，暴露的神經(jīng)組織均用石蠟油覆蓋。用熱反饋式電熱毯使大鼠肛溫維持在37.0 ℃-37.5 ℃。脊髓背角C纖維誘發(fā)電位的記錄方法如文獻(xiàn)所述[8,9]。簡單地說, 通過氯化銀電極電刺激游離的左側(cè)坐骨神經(jīng)以誘發(fā)脊髓場電位，鎢絲微電極(電阻0.5-1 MΩ)進(jìn)行細(xì)胞外記錄，用電控的微量推進(jìn)器(Narishige Scientific Instrument Laboratory)將電極深度控制在脊髓表面下100-500 μm。用數(shù)/模轉(zhuǎn)換器(ADC-42，PICO)以10 kHz的速度處理和儲存數(shù)據(jù)。以C纖維誘發(fā)電位的幅度作為參數(shù)。單方波(10-20 V，0.5 ms，1 min)刺激分離的左側(cè)坐骨神經(jīng)誘發(fā)脊髓背角場電位，高頻刺激(40 V，0.5 ms，100 Hz，持續(xù)1 s，間隔10 s，4次)誘導(dǎo)C纖維誘發(fā)電位LTP。坐骨神經(jīng)的刺激點(diǎn)到脊髓背角的記錄點(diǎn)約為11 cm。

2.2Western blotting 分離刺激同側(cè)腰4-腰5(L4-L5)脊髓背角組織,在培養(yǎng)皿中去除軟脊膜和殘留血塊,將組織迅速放入液氮罐中(10 s), 隨后將組織按重量加入Tris 裂解緩沖液[超純水 2.8 mL, 1.0 mol/L Tris (pH 6.8) 1 mL, 10% SDS 6 mL, 2% β-巰基乙醇 0.2 mL; 10 mL配方] 中,加入蛋白酶抑制劑混合物cocktail (1∶1 000; Roche Molecular Biochemicals) 和磷酸酶抑制劑 (1∶1 000), 冰上勻漿，超聲破碎, 低溫離心。 取上層含組織蛋白的液體分裝保存在-80 ℃。用Micro-BAC法測定蛋白濃度。取等量蛋白樣品(50 μg)加入1/10體積的上樣緩沖液,混勻沸水浴10 min變性。隨后加入SDS-PAGE膠孔中,電泳分離蛋白,電轉(zhuǎn)至PVDF膜上。常溫下5%封閉液封閉PVDF膜2 min,Ⅰ抗孵育,4 ℃搖床過夜。洗脫3次,每次15 min,常溫下Ⅱ抗孵育2 h,洗脫3次,每次15 min,ECL顯色,曝光。

2.3免疫組織化學(xué) 取出誘導(dǎo)LTP后不同時(shí)段或不同藥物處理后的大鼠，首先經(jīng)主動(dòng)脈快速灌注4 ℃肝素化的生理鹽水300 mL，然后用4 %多聚甲醛300 mL灌注30 min，解剖動(dòng)物取出腰膨大脊髓后，再放入4 %的多聚甲醛溶液中后固定3 h，隨后轉(zhuǎn)入30 %蔗糖中脫水2 d。標(biāo)本經(jīng)蔗糖脫水后進(jìn)行冰凍切片(Leica CM1900)并收集于含有0.01 mol/L PBS 的24 孔板內(nèi)，4 ℃短暫保存。脊髓為橫切片，厚度為25 μm。收集脊髓冰凍切片立即用0.01 mol/L PBS 洗3 次，每次5 min，然后室溫下用封閉液作用于切片，以封閉非特異性結(jié)合位點(diǎn)。1 h后吸去封閉液并加入兔抗-大鼠 p-SFKs 抗體(1∶500)，4 ℃搖床下作用48 h 后，吸去Ⅰ抗，用0.01 mol/L PBS 洗3 次，加入標(biāo)記CY3 的驢抗兔Ⅱ抗(1∶200)，避光室溫下作用1 h，再用0.01 mol/L PBS 洗3 次，隨機(jī)挑選切片貼于載玻片上，立即于熒光顯微鏡(Olympus IX71)下觀察并拍照。對于進(jìn)行免疫熒光雙染的脊髓切片，在封閉液處理切片后，立即加入抗-p-SFKs(1∶500)與脊髓細(xì)胞標(biāo)志物(抗-NeuN、抗-GFAP或抗- OX-42)抗體的混合物，常溫作用2 h。切片經(jīng)以上抗體雙標(biāo)記后，用0.01 mol/L PBS 洗3 次，然后加入FITC 和CY3標(biāo)記的2種Ⅱ抗(1∶200)，避光室溫下反應(yīng)1 h，再用0.01 mol/L PBS 洗片，隨機(jī)挑選切片貼片后，立即于熒光顯微鏡下觀察并拍照。

3統(tǒng)計(jì)學(xué)處理

結(jié) 果

1SFKs的抑制劑對脊髓LTP誘導(dǎo)的影響

PP2(100 μmol/L, 200 μL)不影響C纖維基礎(chǔ)電位,但是可以將高頻刺激誘導(dǎo)的LTP逆轉(zhuǎn)為長時(shí)程抑制(long-term depression, LTD),即脊髓LTP不能被誘導(dǎo)。而用PP2的無活性結(jié)構(gòu)類似物PP3作用后,高頻刺激(high-frequency stimulation, HFS)坐骨神經(jīng)依然可以誘導(dǎo)出脊髓LTP,見圖1A。SFKs的另一種廣譜抑制劑SU6656也得到相同的結(jié)果，見圖1B。以上結(jié)果表示SFKs的激活是脊髓LTP誘導(dǎo)的必要條件。

Figure 1. High-frequency stimulation(HFS) induce LTD in animals pretreated with SFKs inhibitor PP2 or SU6656. A: PP2 (100 μmol/L, filled circle) enabled HFS to induce LTD,but its inactive form PP3 (100 μmol/L, filled triangle) enabled HFS to induce LTP. The same dose of PP2 did not affect baseline of C-fiber responses (open circle). B: pretreatment with SU6656 (200 μmol/L), which did not affect basal synaptic transmission (open circle), also reversed the effect of HFS (closed circle).

2高頻刺激誘導(dǎo)脊髓LTP后,脊髓背角SFKs磷酸化表達(dá)明顯上升

結(jié)果如圖2所示,與對照組相比,高頻刺激誘導(dǎo)脊髓LTP后15 min,脊髓背角p-SFKs表達(dá)大量增加 (Plt;0.05)。一直持續(xù)到誘導(dǎo)LTP后60 min恢復(fù)到對照組水平(Pgt;0.05).而SFKs蛋白總量保持不變。

Figure 2. The Src-family kinases were activated after LTP induction in spinal doral horn. A: the bands showed the phosphorylated and total SFKs and β-actin from protein samples of ipsilateral spinal dorsal horn (L4-L5) 15, 30, 60 and 180 min after LTP induced by HFS. In control animals C-fiber responses were recorded but no HFS was conducted. B: the bar chart showed the p-SFKs levels that were normalized by total SFKs levels and expressed as percentages of their controls±s.n=3**Plt;0.01 vs control.

3高頻刺激誘導(dǎo)脊髓LTP后,脊髓背角SFKs磷酸化表達(dá)僅存在于小膠質(zhì)細(xì)胞中

結(jié)果如圖3所示,脊髓背角p-SFKs和小膠質(zhì)細(xì)胞的標(biāo)記物OX-42 (圖3E)共存而不是和神經(jīng)元的標(biāo)記物NeuN (圖3D)或星形膠質(zhì)細(xì)胞的標(biāo)記物GFAP (圖3F) 共存。這表明高頻刺激誘導(dǎo)脊髓LTP后,脊髓背角SFKs的激活僅僅存在于小膠質(zhì)細(xì)胞中,這與已經(jīng)報(bào)道的神經(jīng)損傷引起脊髓小膠質(zhì)細(xì)胞中SFKs的大量激活是一致的。為驗(yàn)證抗體特異性,我們?nèi)〔糠智衅隽嗣庖邿晒鈫稳?，結(jié)果LTP組(圖3C)脊髓背角p-SFKs的表達(dá)明顯高于對照組(圖3B) p-SFKs的表達(dá)。

Figure 3. The activated Src-family kinases were exclusively expressed in spinal microglia after LTP induction. A: the photograph showed no signal was detected in negative control experiments, in which only secondary antibody but no primary antibody was added (n=2). B-C: the photographs showed the expression of p-SFKs in spinal dorsal horn 15 min after HFS(C) and that in control animals(B). D-F: the photographs showed that p-SFKs (E, red) were co-localized with OX-42 (microglia marker, E, green), but without NeuN (neuron marker, D, green) or GFAP (astrocytes marker, F, green). The yellow cells indicated double-labeled cells. Scale bar = 100 μm (A-F).

討 論

本實(shí)驗(yàn)結(jié)果顯示,抑制SFKs可以完全阻斷脊髓LTP的誘導(dǎo),甚至將高頻刺激誘導(dǎo)的LTP逆轉(zhuǎn)為LTD。脊髓LTP誘導(dǎo)后15 min開始,刺激同側(cè)脊髓背角中SFKs的磷酸化表達(dá)明顯增加,維持到LTP誘導(dǎo)后60 min恢復(fù)到對照水平,這些大量表達(dá)的磷酸化SFKs僅僅存在于脊髓小膠質(zhì)細(xì)胞中。因此SFKs的激活是誘導(dǎo)脊髓背角C纖維誘發(fā)電位LTP的必要條件。

大量文獻(xiàn)報(bào)道，SFKs 在中樞神經(jīng)系統(tǒng)的發(fā)育過程中是各種信號途徑的交會點(diǎn)，在學(xué)習(xí)和記憶等生理可塑性過程以及癲癇和神經(jīng)退行性變等病理可塑性過程中起關(guān)鍵作用[10,11]。在海馬,SFKs表達(dá)在突觸后致密區(qū)(post synaptic density, PSD),在海馬LTP誘導(dǎo)后1-5 min就大量激活[12],抑制了SFKs可以阻斷LTP的誘導(dǎo),但不能逆轉(zhuǎn)為LTD。而灌注Src可以增強(qiáng)AMPA(alpha-amino-3-hydroxy-5-methl-4-isoxalzole propionic acid)受體介導(dǎo)的電流,后者是依賴于鈣離子流入NMDA(N-methyl-D-aspartate)受體通道的。在脊髓背角，最近有研究表明腰5 脊神經(jīng)損傷后1d，脊髓背角SFKs 磷酸化水平顯著升高，持續(xù)至少14 d。SFKs 抑制劑PP2 抑制神經(jīng)損傷引起的機(jī)械痛敏。與海馬不同，腰5脊神經(jīng)損傷引起的SFKs 激活僅發(fā)生在脊髓小膠質(zhì)細(xì)胞，而在脊髓神經(jīng)元、星形膠質(zhì)細(xì)胞及腰4、腰5 背根神經(jīng)節(jié)均未檢出SFKs 的磷酸化[13]。

我們第二部分的結(jié)果表明,與正常大鼠和進(jìn)行電生理記錄而未給高頻刺激的大鼠相比,高頻刺激誘導(dǎo)脊髓LTP后非常短的時(shí)間內(nèi)(15 min)脊髓背角磷酸化的SFKs表達(dá)明顯增加(300 %),可以維持到LTP誘導(dǎo)后60 min。而免疫熒光雙染結(jié)果又證明這種大量表達(dá)的p-SFKs僅僅存在于小膠質(zhì)細(xì)胞而非神經(jīng)元或者星形膠質(zhì)細(xì)胞中。這些結(jié)果和已經(jīng)報(bào)道的神經(jīng)損傷后脊髓背角磷酸化SFKs表達(dá)增加,并且只存在于小膠質(zhì)細(xì)胞中的結(jié)果是一致的。不同的是外周神經(jīng)機(jī)械損傷后脊髓中p-SFKs表達(dá)的時(shí)間可以持續(xù)到術(shù)后14 d, 而我們Western blotting的結(jié)果發(fā)現(xiàn)高頻刺激坐骨神經(jīng)誘導(dǎo)LTP后15 min時(shí)脊髓背角p-SFKs明顯增加,到60 min時(shí)恢復(fù)到對照水平,這可能是機(jī)械切斷外周神經(jīng)和高頻刺激坐骨神經(jīng)這兩種刺激方式不同造成的。根據(jù)現(xiàn)有文獻(xiàn)報(bào)道,小膠質(zhì)細(xì)胞的激活在病理性疼痛的產(chǎn)生中起重要作用,而病理性疼痛的維持需要星形膠質(zhì)細(xì)胞的激活。而我們Western blotting的結(jié)果也證明高頻刺激誘導(dǎo)脊髓LTP后15 min-60 min,脊髓背角SFKs大量激活,說明SFKs僅僅在LTP的早期誘導(dǎo)階段起作用,其維持階段需要激活另外的信號通路最終活化NF-kappa B導(dǎo)致新的蛋白質(zhì)合成。LTP誘導(dǎo)階段需要激活小膠質(zhì)細(xì)胞中的SFKs, LTP誘導(dǎo)后的維持階段可能需要星形膠質(zhì)細(xì)胞和突觸后神經(jīng)元的共同參與。

綜合上述結(jié)果并結(jié)合已有研究,我們認(rèn)為脊髓LTP的誘導(dǎo)和維持是一個(gè)復(fù)雜的過程,需要多種因素參與；脊髓小膠質(zhì)細(xì)胞中SFKs的激活在脊髓LTP的誘導(dǎo)中起著不可缺少的作用；晚期LTP的維持則需要新蛋白質(zhì)的合成；以小膠質(zhì)細(xì)胞及其中SFKs為靶點(diǎn)的藥物將有助于治療病理性疼痛。

[1] Liu XG, Sandkühler J. Long-term potentiation of C-fiber-evoked potentials in the rat spinal dorsal horn is prevented by spinalN-methyl-D-aspartic acid receptor blockage [J]. Neurosci Lett, 1995, 191(1-2): 43-46.

[2] Zhang HM, Zhou LJ, Hu XD, et al. Acute nerve injury induces long-term potentiation of C-fiber evoked field potentials in spinal dorsal horn of intact rat [J]. Acta Physiologica Sinica, 2004, 56(5): 591-596.

[3] Klein T, Magerl W, Hopf HC, et al. Perceptual correlates of nociceptive long-term potentiation and long-term depression in humans [J]. J Neurosci, 2004, 24(4): 964-971.

[4] Ji RR, Kohno T, Moore KA, et al. Central sensitization and LTP: do pain and memory share similar mechanisms? [J]. Trends Neurosci, 2003, 26(12): 696-705.

[5] Willis WD. Long-term potentiation in spinothalamic neurons [J]. Brain Res Rev, 2002, 40(1-3): 202-214.

[6] Kuo WL, Chung KC, Rosner MR. Differentiation of central nervous system neuronal cell by fibroblast-derived growth factor requires at least two signaling pathways: role of Ras and Src [J]. Mol Cell Biol, 1997, 17(8): 4633-4643.

[7] Hoffman-Kim D, Kerner JA, Chen A, et al. pp60c-srcis a negative regulator of laminin-1 -mediated neurite outgrowth in chick sensory neurons [J]. Mol Cell Neurosci, 2002, 21(1): 81-93.

[8] Liu XG, Sandk ( (hler J. Characterization of long-term potentiation of C-fiber-evoked potentials in spinal dorsal horn of adult rat: essential role of NK1 and NK2 receptors [J]. J Neurophysiol, 1997, 78(4): 1973-1982.

[9] Liu XG, Sandk ühler J. Activation of spinalN-methyl-D-aspartate or neurokinin receptors induces long-term potentiation of spinal C-fibre-evoked potentials in spinalized rats [J]. Neuroscience, 1998, 86(4): 1209-1216.

[10]Purcell AL, Carew TJ. Tyrosine kinases, synaptic plasticity and memory: insights from vertebrates and invertebrates [J]. Trends Neurosci, 2003, 26(11): 625-630.

[11]Kalia LV, Gingrich JR, Salter MW. Src in synaptic transmission and plasticity [J]. Oncogene, 2004, 23(48): 8007-8016.

[12]Soderling TR, Derkach VA. Postsynaptic protein phosphorylation and LTP [J]. Trends Neurosci, 2000, 23(2): 75-80.

[13]Katsura H, Obata K, Mizushima T, et al. Activation of Src-family kinases in spinal microglia contributes to mechanical hypersensitivity after nerve injury [J]. J Neurosci, 2006, 26(34): 8680-8690.

TheroleofSrc-familykinasesinlong-termpotentiationinratspinaldorsalhorn

ZHONG Yi1, HUANG Yang-liang2, XU Ji-de1

(1DepartmentofPhysiology,GuangzhouMedicalCollege,Guangzhou510182,China;2DepartmentofSpineSurgery,TheFirstAffiliatedHospital,SunYet-senUniversity,Guangzhou510700,China.E-mail:victoria0720@126.com)

AIM: To investigate the role of Src-family kinases (SFKs) in long-term potentiation (LTP) of C-fiber-evoked potentials in rat spinal dorsal horn and the underlying mechanism.METHODSThe effect of SFKs inhibitors on spinal LTP induced by high-frequency stimulation of sciatic nerve was determined by electrophysiological method. The levels of phosphorylated SFKs (p-SFKs) in spinal dorsal horn were measured by Western blotting at different time points after LTP induction. The cell types that expressed p-SFKs following LTP induction were determined by double-labeled immunofluorescence staining.RESULTSElectrophysiological data revealed that pretreatment with SFKs inhibitors (PP2 or SU6656) enabled high-frequency stimulation to induce long-term depression (LTD) rather than LTP in spinal dorsal horn. Western blotting analysis showed that the level of p-SFKs in ipsilateral spinal dorsal horn increased at 15 min after LTP induction. Double-labeled immunofluorescence staining demonstrated that p-SFKs were highly restricted to spinal microglia.CONCLUSIONSFKs in microglia play a critical role in the induction of LTP in spinal dorsal horn. Inhibition of SFKs and the downstream molecules may be helpful for curing neuropathic pain.

Src-family kinases; Long-term potentiation; Pain; Spinal dorsal horn

1000-4718(2011)04-0727-05

R338

A

10.3969/j.issn.1000-4718.2011.04.020

2010-11-05

2011-02-25

△通訊作者 Tel: 020-81340199; E-mail: victoria0720@126.com