TLR4信号通路在阿片源性痛觉过敏脊髓小胶质细胞活化中的作用及机制

mu-Opioid receptor agonists represent the gold standard for the treatment of severe pain but may paradoxically also enhance pain sensitivity, that is, lead to opioid-induced hyperalgesia (OIH).OIH is correlated with all kinds of opioids including morphine, fentanyl and remifentanil. But the mechanism involved in OIH is still unclear. Studies have showed that morphine can induced signifcant spinal glia activation but not include fentanyl or remifentanil.Glial cells and TLR4 pathway are recognized as performing important role in hyperalgesia induced by morphine.However, whether glia hyperactivity is essential in OIH induced by other opioids has not been verified. Opioid receptors are both expressed in spinal glia and neuron cells.But how glial cells were activated free of antinociceptive stumuli,through which way did opioid agonists activate glial cell are still unknown. In our previous study, we have showed that both morphine and fentanyl can cause significant hperalgesia after drug termination. Morphine could induced spinal glia fibrillary acidic proteion(GFAP, present exclusively in astrocytes) overexpression. We hypothsis that glial activation and TLR4 plays an differert role in vatiou kinds of opioids such as morphine and remifenanil. The present study includes two parts. Firstly,in vitro stuty, we set up OIH animal models using morphine, fentanyl and remifentanil,and nextly detect the spinal expression and levels of GFAP,OX-42 and TLR4,thus and clarify whether glia activation is essential in OIH.We also use the TLR4 agonist LPS and antagnist LPS-RS to detect the effect of TLR4 pathway in OIH and glial activation. In vivo study,we try to detect the differences in GFAP, OX-42, TLR4, expression between various drugs and TLR agonist or antagnist in glial cultures.Through above ways, we try to clarify the pathway how glial cells were activated and the importance of TLR4 pathway in activation of glia in OIH.

阿片类药物长期大剂量应用可导致机体对伤害性刺激的敏感性增加，此现象称阿片源性痛觉过敏(OIH),各种阿片类药物均可诱发OIH，脊髓小胶质细胞(glia)的激活参与痛觉敏化的发生,Toll样受体4(TLR4)及其信号通路可能在阿片药物介导的小胶质活化发挥重要的桥梁作用。课题组的前期研究证实吗啡、芬太尼长期或大量应用可诱导OIH现象,并且吗啡还可激活脊髓星形胶质细胞激活。最新的研究提示瑞芬太尼与吗啡诱导的OIH内在机制不同，而芬太尼衍生物是否可通过小胶质细胞膜上的TLR4受体，诱导glia活化？目前尚不清楚。本研究首先通过免疫组化，蛋白印迹测定OIH模型动物脊髓GFAP，OX-42以及TLR4及下游信号通路的改变，明确OIH与小胶质细胞活化关系，再进一步应用TLR4激动剂和拮抗剂，明确TLR4通路是激活glia的重要机制，为揭示阿片类药物致中枢敏化的机制以及寻找新的药物靶点提供实验依据。

临床研究部分.在《静脉麻醉药物在亲属活体肾移植患者中的药效和药代动力学研究》中。实验第一部分为《右美托咪定用于终末期肾功能衰竭行肾移植手术的安全性评价》，本研究将右美托咪定用于ESRD拟行亲体肾移植患者的麻醉，评价其安全性并观察对患者苏醒期躁动、术后尿量及肾功能的影响。。.实验第二部分，纳入ESRD拟行肾移植的患者和拟施手术需要行全身麻醉的正常人群作为对照，采用靶控输注丙泊酚作为麻醉诱导和维持的方法，通过监测患者麻醉深度（BIS，脑电双频指数）；并抽取血样采用高效液相法测定丙泊酚的实际血药浓度，评价靶控输注丙泊酚在肾移植患者中应用的安全及有效性。研究成果及科学意义：①右美托咪定可安全用于ESRD拟行亲体肾移植患者的麻醉。②靶控输注丙泊酚可安全有效用于ESRD患者肾移植手术的麻醉。.关于这方面的研究，撰写论文3篇，发表2篇，待发表论文1篇。.基础研究部分.TLR样受体在人体及大鼠疼痛阈值改变及机制的研究.本研究分为两部分，第一部分为人体试验。针对临床常见的急性疼痛（骨折）、慢性疼痛患者（慢性骨关节炎）以及癌性疼痛患者，对其疼痛阈值和焦虑状态进行痛行为学测定，并通过量表评价患者的焦虑状态，测定患者外周血白细胞介素-6，C反应蛋白以及外周血单核细胞TLR4受体的表达情况，比较不同类型疼痛患者之间疼痛阈值的以及TLR4受体的差异，为疼痛治疗提供理论和实践依据，并探讨TLR4受体在疼痛阈值改变中的意义。.第二部分为动物实验，针对瑞芬太尼诱导的切口痛大鼠痛觉过敏中TLR-4发挥的作用及机制进行研究。通过建立切口痛模型，模拟临床手术切口疼痛，再应用瑞芬太尼输注造成阿片源性的痛觉过敏模型，在测定大鼠脊髓、脑内以及外周血单核细胞TLR4受体的表达水平，评价TLR4受体在该类型疼痛模型中的意义。.研究结果及科学意义： TLR4样受体在疼痛阈值降低的机制中发挥重要作用。可能与外周炎症反应炎性因子的增高以及脊髓胶质细胞的活化有关。.关于这方面的研究，撰写论文2篇，并培养硕士研究生2名。