老年大鼠脊髓小胶质细胞分选新方法及功能特征观察*

目的: 建立分离纯化老年大鼠小胶质细胞的改良方法,并初步观察老年大鼠脊髓小胶质细胞的生物学特性。方法: 以年轻SD大鼠(2月龄)为对照组,采用胰酶、胰酶替代物和机械网搓法等不同的制备方法,制备大鼠小胶质细胞的单细胞悬液,通过检测细胞纯度、存活率,观察细胞形态特征,分析细胞的炎性功能特征等,确定老年大鼠(20月龄)小胶质细胞的分离纯化方法,观察老年大鼠脊髓小胶质细胞功能特征。结果: 胰酶消化所得细胞的存活率低(年轻大鼠83%,老年大鼠60%);机械网搓法虽得到的存活率较高(95%),但是细胞获取率最低(年轻大鼠((0.207±0.020)×10⁶,老年大鼠(0.243±0.023)×10⁶);采用胰酶替代物解离、密度梯度离心方法分选出的老年大鼠脊髓小胶质细胞数量多、活性好、存活率高,细胞纯度可达85%以上,我们采用此方法分选纯化不同年龄大鼠脊髓小胶质细胞,与年轻大鼠相比,老年鼠脊髓组织量大,所需消化液多,但消化时间缩短;与年轻大鼠小胶质细胞相比,老年大鼠脊髓小胶质细胞其胞体较大较圆,突起少且粗短,形态上偏向于激活状态,老年大鼠小胶质细胞促炎因子IL-1β表达降低(P＜0.05),而抗炎因子IL-10(P＜0.01)表达升高。结论: 成功建立胰酶替代物解离结合密度梯度离心法从大鼠脊髓组织中分离纯化小胶质细胞,老年大鼠脊髓内小胶质细胞整体表现出抗炎表型。     

淋巴细胞抑制剂能够抑制脊髓小胶质细胞活化并缓解CCI大鼠的热痛敏

近年有观点认为,进入慢性疼痛患者外周神经系统中的淋巴细胞过度活化,是疼痛经久不愈的原因之一。但是根据疼痛发生的原理,伤害性刺激是在脊髓进行整合的。那么,淋巴细胞是否可以影响到脊髓的功能状态呢？我们认为,淋巴细胞可以作用于脊髓小胶质细胞,从而影响疼痛知觉的产生。为了验证这个观点,我们首先制作大鼠坐骨神经慢性压榨性损伤（CCI）模型。     

环氧合酶-1抑制剂对术后疼痛大鼠脊髓中ERK表达的影响

目的：探讨术前鞘内注射选择性环氧合酶-1（cyclooxygenase-1）抑制剂SC-560对术后疼痛大鼠机械痛觉超敏以及脊髓中磷酸化ERK（p-ERK）蛋白表达的影响。方法：采用术后疼痛模型,于术后1h通过观察机械缩足反射阈值（MwT）测定术后痛觉超敏情况、免疫组化染色和免疫印迹检测脊髓中p-ERK表达的变化。结果：①术后疼痛大鼠术后1h出现明显痛觉超敏反应,术前鞘内注射SC-560 100μg可以明显抑制术后痛觉超敏;②术后1h大鼠腰段脊髓背角浅层p-ERK免疫组化染色阳性细胞数较正常对照组明显增加,鞘内注射SC-560可明显减少p-ERK阳性细胞数（P〈0．01）;术后1h大鼠腰段脊髓Western blot检测,p-ERK1／2蛋白的表达较正常大鼠均明显增加,鞘内注射9G560可以抑制p-ERK1／2蛋白的表达。结论：鞘内注射CO3X-1抑制剂可以抑制术后疼痛大鼠术后痛觉超敏反应,脊髓水平Ⅱ水可能介导其上述作用。     

丁苯酞对睡眠剥夺大鼠额叶小胶质细胞活化的影响

目的:探讨丁苯酞对慢性睡眠剥夺后大鼠脑部额叶小胶质细胞活化及炎症因子的影响。方法:本实验共分为4组(n=8):空白对照组、大平台对照组、慢性睡眠剥夺组、丁苯酞干预组。慢性睡眠剥夺组和丁苯酞干预组采用改良多平台睡眠剥夺法建立大鼠慢性睡眠剥夺模型,对大鼠进行每日18 h,连续28 d的睡眠剥夺。在这28d内,空白对照组大鼠不进行睡眠干预,大平台对照组大鼠放于大平台箱内。丁苯酞干预组在睡眠剥夺28 d结束后按100 mg/kg腹腔注射丁苯酞针剂,每日1次,共14 d,其他组大鼠在这14 d内腹腔注射同样剂量的生理盐水。腹腔注射结束后各组大鼠取脑组织,免疫组化检测额叶皮质离子钙接头分子(Iba-1)阳性细胞并计数,Western blot检测额叶诱导型一氧化氮合成酶(iNOS)、精氨酸酶1(Arg1)表达,实时定量PCR检测额叶白介素-1(IL-1) mRNA、IL-6 mRNA、肿瘤坏死因子-α(TNF-α) mRNA。结果:与空白对照组、大平台对照组比较,慢性睡眠剥夺组额叶Iba-1阳性细胞体积增大伴细胞突起增多,且细胞数增加(P均<0.05),iNOS和IL-1 mRNA、IL-6...     

间充质干细胞来源外泌体对脑内小胶质细胞极化和炎症因子释放的影响及机制研究

摘要 目的：探究间充质干细胞外泌体对脑内小胶质细胞极化和炎症因子释放的影响及其机制。方法：收集体外培养的间充质干细胞上清，超高速冷冻离心获取外泌体。采用纳米颗粒系统和透射电子显微镜分别检测外泌体粒径大小、形态结构和功能完整性。通过免疫荧光、ELISA和细胞流式等方式检测LPS刺激下，外泌体对BV2细胞的表型极化和炎症因子释放的影响。采用Western Blot法检测间充质干细胞外泌体对BV2细胞JAK1/STAT3通路活化的影响。结果：（1）间充质干细胞分泌的外泌体粒径大小主要介于40-100 nm，透射电镜显示外泌体形态呈典型膜性"杯盘"状结构；（2）流式结果表明，相比于对照组，LPS组能显著激活M1型小胶质细胞表面标志物CD11b和M2型小胶质细胞表面标志物CD206的表达，而经外泌体处理，CD11b的表达显著被抑制，CD206显著升高。同时ELISA结果证实，相比于LPS组，外泌体组分泌的促炎症因子（IL-1β、IL-6）和NO水平显著降低（P<0.05），抗炎因子（IL-10）显著升高 (P<0.05)；（3）间充质干细胞外泌体显著提高了BV2细胞JAK1/STAT3通路的磷酸化水平。结论：间充质干细胞外泌体通过激活JAK1/STAT3通路有效促进脑内小胶质细胞M1型向M2型极化。     

鞘内注射孤啡肽对大鼠足底注入蜜蜂毒诱致长时程自发痛、痛敏和炎症的不同效果

为进一步了解孤啡肽在脊髓水平是否具有抗伤害及抗炎作用,本实验在具有多种痛行为表现的蜜蜂毒模型上观察了鞘内注射孤啡肽对大鼠一侧后足底注入蜜蜂毒所诱致的同侧自发缩足反射、原发热和机械性痛敏以及注射部位炎症反应的影响,同时观察了新的高选择性孤啡肽受体拮抗剂CompB的作用.结果表明与生理盐水对照组比较,鞘内注射孤啡肽(3、10、30 nmol/10μl)对蜜蜂毒诱发的自发缩足反射次数的抑制作用随剂量提高而增大,抑制率分别为37±7,43±6and57±11%(三个剂量vs对照,P＜0.05);而对蜜蜂毒诱发的注射部位炎症反应(爪体积、爪背腹厚度和蛋白渗出的增加)无显著影响.CompB(30 nmo1)可完全翻转10 nmol孤啡肽对自发缩足反射的抑制作用.鞘内单次或重复注射孤啡肽(10 nmol/10μl)对蜜蜂毒诱致的原发性热和机械性痛敏的发生和维持均无作用.本实验结果提示,外源性孤啡肽在脊髓通过孤啡肽受体的介导产生一定的镇痛作用,但是它可能仅对持续性自发痛有抑制作用,而对热和机械性痛敏及炎症反应均无影响.     

甲醛炎性痛及痛过敏诱导大鼠脊髓后角环氧化酶-2表达的变化

目的:观察甲醛炎性痛及痛过敏过程中脊髓后角环氧化酶-2 (COX-2)表达的变化及其时间特征.方法:采用大鼠甲醛疼痛模型,用免疫组化法观察脊髓COX-2表达的变化.结果:与对照组相比,注射甲醛后4 h,1 d及3 d组脊髓后角Ⅰ-Ⅵ板层COX-2免疫反应阳性细胞的数目及染色深度均显著增加,以1 d组增加最为明显.结论:脊髓后角COX-2参与甲醛炎性痛及痛过敏.     

Direct evidence for spinal cord microglia in the development of a neuropathic pain-like state in mice

The present study was undertaken to further investigate the role of glial cells in the development of the neuropathic pain-like state induced by sciatic nerve ligation in mice. At 7 days after sciatic nerve ligation, the immunoreactivities (IRs) of the specific astrocyte marker glial fibrillary acidic protein (GFAP) and the specific microglial marker OX-42, but not the specific oligodendrocyte marker O4, were increased on the ipsilateral side of the spinal cord dorsal horn in nerve-ligated mice compared with that on the contralateral side. Furthermore, a single intrathecal injection of activated spinal cord microglia, but not astrocytes, caused thermal hyperalgesia in naive mice. Furthermore, 5-bromo-2'-deoxyuridine (BrdU)-positive cells on the ipsilateral dorsal horn of the spinal cord were significantly increased at 7 days after nerve ligation and were highly co-localized with another microglia marker, ionized calcium-binding adaptor molecule 1 (Iba1), but neither with GFAP nor a specific neural nuclei marker, NeuN, in the spinal dorsal horn of nerve-ligated mice. The present data strongly support the idea that spinal cord astrocytes and microglia are activated under the neuropathic pain-like state, and that the proliferated and activated microglia directly contribute to the development of a neuropathic pain-like state in mice.     

MK—801降低炎性痛在鼠脊髓NOS表达和NO含量

用NADPH-d组织化学法,观察鞘内注射NMDA受体拮抗剂MK-801对大鼠右后掌皮下注射甲醛诱发的炎症性痛及痛过敏过程中脊髓后角一氧化氮合酶(NOS)表达的影响,同时测定一氧化氮(NO)代谢终产物     

Activation of p38 mitogen-activated protein kinase in spinal microglia is a critical link in inflammation-induced spinal pain processing

We examined the effect of p38 mitogen-activated protein kinase (MAPK) inhibitors in models of nociception and correlated this effect with localization and expression levels of p38 MAPK in spinal cord. There was a rapid increase in phosphorylated p38 MAPK in spinal cord following intrathecal administration of substance P or intradermal injection of formalin. Immunocytochemistry revealed that phosphorylated p38 MAPK-immunoreactive cells were predominantly present in laminae I-IV of the dorsal horn. Double-staining with markers for neurons, microglia, astrocytes and oligodendrocytes unexpectedly revealed co-localization with microglia but not with neurons or other glia. Pretreatment with p38 MAPK inhibitors (SB20358 or SD-282) had no effect on acute thermal thresholds. However, they attenuated hyperalgesia in several nociceptive models associated with spinal sensitization including direct spinal activation (intrathecal substance P) and peripheral tissue inflammation (intraplantar formalin or carrageenan). Spinal sensitization, manifested by enhanced expression of cyclo-oxygenase-2 and inflammation-induced appearance of Fos-positive neurons, was blocked by pretreatment, but not post-treatment, with p38 MAPK inhibitors. Taken together, these results indicate that spinal p38 MAPK is involved in inflammation-induced pain and that activated spinal microglia play a direct role in spinal nociceptive processing.     

早期干预ERK在脊髓的激活可阻断外周神经损伤引起的神经病理性疼痛的发生

本文采用大鼠坐骨神经慢性压迫损伤引起的神经病理痛模型,研究脊髓背角细胞外信号调节激酶(extracellular signal-regulatedkinase,ERK)在外周神经损伤引起的神经病理疼痛发生中的作用.结果显示,单侧坐骨神经压迫性损伤后1天,大鼠损伤侧脊髓背角ERK的磷酸化(激活)水平显著上调,其下游转录因...