【作者】 曾燕；

【导师】 施静；

【作者基本信息】 华中科技大学 ， 中西医结合基础， 2007， 博士

【摘要】 第一部分持续去极化诱导背根神经节神经元间ATP-P2Y系统依赖的钙振荡扩布虽然神经元之间最主要的通讯方式是化学突触,但是电突触和非突触性化学传递也占据着重要地位。可在背根神经节(dosal root ganglion, DRG)内的初级神经元之间因为卫星细胞的分隔,即缺少化学突触也无电突触,而大量的研究发现却表明节内的神经细胞间能相互影响,即刺激诱导一个神经细胞兴奋,另一个邻近的细胞也被动发生兴奋性改变,这种现象被命名为“交叉兴奋”(cross excitation),而产生这种现象的机制还只是在猜测之中。以视神经节为材料的研究又发现,节内神经细胞兴奋释放的活性物质通过它相应的受体能诱导细胞间通讯,然而对这种胞间通讯模式机制的确定,以及在其他节内神经细胞间寻找更多的支持显得非常必要。在本研究中,我们在培养的DRG神经元间发现了一个由ATP介导的非突触性化学传递模式。单个DRG神经元持续去极化激起这个细胞的胞内钙振荡性升高,持续性的钠离子内流,以及接下来细胞体的ATP释放并向周围扩散最终诱发邻近未受刺激的被动细胞内规则的钙振荡。钙振荡和钠离子内流均能被TTX抑制,但不受细胞外钙离子浓度的影响,且在无钙溶液中仍能发生,表明钠离子内流介导的细胞膜去极化以及胞内钙释放是钙振荡产生的原因。进一步研究发现,钙振荡产生的源在IP3受体引起的内质网钙库的钙释放,而不由ryanodine受体介导。采用ATP实时成像跟踪了ATP的释放和扩散,且ATP水解酶彻底瓦解钙振荡扩散,说明神经激活释放了化学物质ATP,它们在DRG神经元间担当着传递信使。我们又发现非选择性P2Y受体抑制剂苏拉明、PLC抑制剂U73122、以及IP3受体拮抗剂2-APB能抑制钙振荡传播,这就表明了介导ATP传播作用的受体途径。为了更充分地描述ATP在这一现象中的作用,外源性ATP被应用到DRG细胞培养中,发现不同浓度的ATP通过P2Y受体在DRG神经元不是引起一过性钙升高,就是诱发钙振荡,10μM—100μM的ATP激起一过性钙升高,100μM—250μM的ATP诱发钙振荡。因此,根据这些数据,我们总结持续去极化刺激引起细胞内钙振荡性释放和ATP释放,胞内钙升高是ATP释放的先决条件,接着释放的ATP通过它的代谢性受体引发了下一个细胞的钙振荡,完成它的传播作用。这是一种特殊的通讯形式,可能是DRG神经元间电兴奋性胞间传递的分子基础。第二部分神经细胞激活控制成纤维细胞间缝隙连接通讯成纤维细胞是非兴奋性细胞,它们高度表达缝隙连接蛋白并形成缝隙连接通讯进行着离子、分子的交流,除了同种细胞间形成广泛的三维结构外,成纤维细胞还与其他类型的细胞形成缝隙连接,比如内皮细胞、肥大细胞、神经细胞等。缝隙连接通讯在成纤维细胞参与的生理和病理过程中起着重要作用,例如参与发育、创伤修复、炎症等过程。在此研究中我们发现DRG神经元和真皮成纤维细胞共培养体系中,神经元活动能抑制成纤维细胞的缝隙连接通讯,它的抑制作用与NMDA谷氨酸受体兴奋以及由它们介导细胞内钙离子升高有关。在共培养的DRG神经元和成纤维细胞样品中,去极化电刺激诱导神经元兴奋后,检测成纤维细胞的染料偶联率,发现成纤维细胞的染料偶联细胞数目减少;并且未受到刺激的共培养体系中成纤维细胞染料偶联率也比单纯成纤维细胞培养的染料偶联率低,这种情况发生在共培养5-6天后;膜片钳记录发现DRG神经元自动发放动作电位和TTX敏感的钠电流;免疫化学检测发现,共培养体系中成纤维细胞最主要的连接蛋白CX43表达减少,可能与连接通讯效率降低有关。但是立即检测给予去极化电刺激的样品中成纤维细胞的主要连接蛋白CX43,发现尽管染料偶联率减少,CX43表达并不减少,考虑缝隙连接通讯效能降低可能还有主要其他机制,进一步研究发现,谷氨酸受体抑制剂MK-801能逆转神经元在缝隙连接通讯上的作用,而且NMDA受体主要介导钙离子内流,细胞内钙升高已经被认为能抑制缝隙连接通讯;接着我们发现NMDA受体和CX43共表达于成纤维细胞膜上,外源性谷氨酸应用能强有力地升高成纤维细胞内钙浓度,并且降低成纤维细胞缝隙连接通讯,这部分作用能被NMDA受体抑制剂MK-801逆转。我们还发现将共培养体系的培养基用于单纯成纤维细胞培养,也导致他们的缝隙连接通讯下降。根据这些观察我们总结去极化刺激导致的神经元激活或者神经元自发活动能释放谷氨酸,通过兴奋NMDA受体,升高细胞内钙抑制成纤维细胞的缝隙连接通讯。因此我们认为除了已经被证明的成纤维细胞对神经元的生长和发育进行调节外,神经元活动也通过其释放的递质对成纤维细胞的通讯功能进行调节。第三部分电针效应在活体裸鼠身上表达的实时监测最近几年,小动物的整体成像已经逐渐发展为一个探测发生在活体动物细胞和分子水平生命活动的强有力的研究工具。在这个研究中,我们用一套整体荧光成像系统,在体检测裸鼠体内可能有的电针效应表达。根据比较动物学研究中在小动物身上确定的几个穴位点,我们尝试在这些穴位点注射低分子量能透过缝隙链接的荧光染料5, 6-carboxyfluorescein和Lucifer yellow以及不能透过缝隙连接的大分子量荧光染料rhodamine dextran,然后进行一系列扫描跟踪荧光染料的扩散,测量它们扩散的速度和范围。结果发现电针能显著诱导低分子荧光染料循着一个方向更快地扩散,而对高分子染料作用不显著,表明电针只诱导能通过缝隙连接的低分子量染料更快的扩散,它可能打开了某一种通道;接下来发现缝隙连接通讯抑制剂acetic acid-Sodium acetate, halothane,或者octanol能阻断电针的这一作用,表明电针的效应与缝隙连接通讯有关。这个研究尝试应用新的研究技术在活体动物身上探测电针效应,并且观察到电针可以通过影响缝隙连接通讯而发挥作用。更多还原

【Abstract】 Section 1 Propagation of Ca2+ oscillation in DRG neurons: the involvement of extracellular ATP and P2Y receptor activationRecently emerging evidence implicates a number of neuroactive substances and their receptors in mediating complex cell-to-cell communications in the ganglia. In the present study, we have characterized the chemical coupling mediated by extracellular ATP in cultured dorsal root ganglion (DRG) neurons by using a real time-imaging of ATP, whole cell patch clamping in conjunction with confocal calcium imaging. Sustained depolarizations of single DRG neurons evoked intracellular Ca2+ concentration ([Ca2+]i) oscillations together with TTX-sensitive persistent Na+ influx, and subsequent ATP-dependent propagation [Ca2+]i oscillations to surrounding passive neighbors. [Ca2+]i oscillations were suppressed by both the Na+ channel blocker TTX and inositol-1,4,5-trisphosphate (IP3) receptor antagonist 2-APB, but not ryanodine. The propagation of [Ca2+]i oscillations was prevented in the presence of the ATP-degrading enzyme, apyrases, and inhibited by suramin, a nonselective P2Y receptor antagonist. In parallel, depolarizing stimuli induced ATP release and diffusion, which was abolished by inhibition of PLC- IP3 pathway or pretreatment with thapsigargin to deplete internal Ca2+ stores. Moreover, exogenous application of ATP to DRG neurons elicited the Ca2+ oscillations involving PLC-IP3 pathway. Taken together, these data demonstrated that sustained membrane depolarization elicited ATP release, acting through a highly sensitive P2Y receptors /IP3-mediated signaling pathway to mediate the propagation of intercellular Ca2+ signaling, which suggest a novel signaling pathway for neuronal communication in DRG.Section 2 Activity-dependent neuronal control of gap-junctional communication in fibroblastsA typical feature of fibroblasts is their high degree of intercellular communication through gap junction channels. Abundant between fibroblasts, gap junction coupling plays important roles in fibroblasts physiology. Here we report that activity-dependent neuronal control of gap junctional communication (GJC) in the rat dermal fibroblasts in vitro is associated to NMDA glutamate receptors-mediated intracellular Ca2+ elevations. Not only excited dorsal root ganglion (DRG) neurons by using depolarizing pulses, which exhibited remarkable action potentials and inward sodium currents, tremendously reduced fibroblast GJC in Neuron/Fibroblast (N/F) cocultures, but also resting DRG neurons, which exhibited frequent spontaneous action potentials and inward sodium currents or so, exerted significant roles on fibroblast GJC and CX43 protein. Both fibroblast GJC and CX43 downregulation were prevented by the blockade of NMDA receptors and decrease of intra- and intercellular Ca2+. Immunocytochemistry showed that the NR1 subunit of the NMDA receptor was colocated at plasma membrane of fibroblasts with CX43. Moreover, glutamate applied to fibroblasts triggered NMDA receptor-dependent intracellular Ca2+ elevations and decline of GJC. These data demonstrate that NMDA receptor activity in fibroblasts contributed to the effect of DRG neurons on fibroblast coupling via downregulation of GJC. Since fibroblasts have been shown to facilitate DRG neurite growth and survival, our findings suggest that neuronal and fibroblasts networks interact actively.Section 3 Real-time whole-body optical imaging of electrical acupuncture responses in intact nude miceWhole-body optical imaging of small animals has emerged as a powerful, user friendly, and high-throughput tool for assaying molecular and cellular processes as they occur in vivo. Here we used a whole-body fluorescence imaging system for the first time in a nude mice model to explore the characteristic responses induced by electrical acupuncture (EA). Nude mice were injected with fluorescent dye at identifiable points similar to the acupoint in Traditional Chinese Medicine for animals. Serial measurements of fluorescence diffusion were performed using in vivo fluorescence macroscopic. EA applied to the acupoint injected with low molecule weight dye evoked a fluorescence channel over a long distance of the fore limb to be coincident with the course of classical meridian. Stimulation applied to the acupoint injected with high molecule weight dyes did not evoke similar fluorescence dye activities. And the activity of low molecular mass dye could be inhibited by gap junction inhibitors including acetic acid-Sodium acetate, halothane, and octanol. This result indicates that the fluorescence dye activity induced by EA is correlated with gap junction communication. Therefore, this study may illustrate objective investigation of EA responses in small animals using a real time whole-body imaging, and evaluate the functional importance on mediating or transmitting EA effectiveness of gap junctions and calcium.更多还原