大鼠面部慢性炎症痛导致三叉神经节表达P2X3神经元表型的转变

目的研究大鼠面部慢性炎症痛与三叉神经节内表达P2X3受体亚型神经元细胞大小和表型变化之间的关系。方法参照Neumann(1996)报道的研究方法,采用大鼠面部皮下注射松节油建立慢性炎症痛模型,用热测痛的方法测定面部皮肤的痛阈值,每天一次,连续测15d。用免疫组织化学技术观察大鼠面部慢性炎症后第5d三叉神经节内感觉神经元P2X3受体的表达。采用体视学的方法测量表达P2X3神经节细胞大小及表型的变化。结果炎症侧大鼠面部痛阈值与对照组相比明显降低,在第5d达到最低值,以后逐渐恢复,第13d开始痛阈恢复正常水平。炎症侧三叉神经节内表达P2X3神经元的平均细胞表面积(721±12μm2)与对照组(616±8μm2)相比明显增大(P<0·01)。进一步观察发现表达P2X3小细胞群(<950μm2)的表面积由炎症前的537±13μm2增加到炎症后的582±15μm2(P<0·05)而且小细胞占总细胞的数量比例由炎症前的42·2±3·2%增加到炎症后的51·8±3·5%(P<0·05);而表达P2X3受体的大细胞(>950μm2)的数量比例由炎症前的6·5±1·9%增加到炎症后的12·8±2·2%(P<0·05)。结论面部慢性炎症痛时,其三叉神经节内表达P2X3受体神经元的表型可发生改变,这可能与面部痛觉过敏和触诱发痛的形成有密切关系。     

P2X3受体在感觉神经节的表达

用免疫组织化学与原位杂交研究P2X3受体在背根神经节,三叉神经节和结状神经节的分布。结果显示：1、原位杂交;在三种感觉神经节中,95%左右的神经节细胞为P2X3mRNA阳性,中、小型神经节细胞的杂交信号一般要比大型的神经节细胞强一些,2、免疫组织化学;免疫组织化学结果与原位杂交结果基本一致。此外,在各神经节内,均显示出许多P2X3免疫阳性神经纤维,在足掌表皮也显示许多P2X3免疫反应阳性纤维,结果提示：P2X3不仅参与机体的痛觉的形成,还可能参与其它感觉,如本体感觉等的形成。     

大鼠初级感觉神经元P2X3受体的表达及其与SP的关系

目的研究在大鼠初级感觉神经元细胞上P2X3受体的表达情况及其与P物质的关系。方法取SD大鼠背根神经节(DRG)和三叉神经节(TG)固定后切片;用抗P2X3受体抗体和抗SP抗体进行免疫组织化学反应,并通过两种不同的显色方法同时进行P2X3受体和SP的双标。结果P2X3免疫反应阳性细胞主要集中在小细胞和中等细胞(其中在TG,P2X3-ir阳性神经元约占整个细胞的24.8%;在DRG约31.7%的神经元是P2X3-ir阳性),并且在DRG和TG细胞上均存在有P2X3受体和SP共存(TG上的双标细胞占P2X3-ir阳性细胞总数的36.26%,DRG上占46.81%)。结论由于ATP门控阳离子通道受体P2X3本身就与伤害性感受的初级传入有关,而它与SP的共存可提示当组织中的ATP释放时可以通过P2X3受体作用于含SP的伤害性感觉神经末梢上,促使SP释放引起痛觉过敏。     

P物质对大鼠DRG神经元胞体膜的作用

本文在大鼠ＤＲＧ神经元标本上应用细胞内记录,以确定ＳＰ对ＤＲＧ细胞的膜反应及其可能的离子机制。实验所测ＤＲＧ细胞静息膜电位为－５８．９±８．２ｍＶ（Ｘ±ＳＥ,ｎ＝８１）。传导速度：Ａ_（α／β）细胞为２０．４±４．８ｍ／ｓ（Ｘ±ＳＥ）,范围１４．１－２８．７ｍ／ｓ（４７／６０）;Ａδ及Ｃ类细胞为９．８±５．２ｍ／ｓ,范围１．２－１３．７ｍ／ｓ（１３／６０）。浴槽滴加ＳＰ（１０￣（－７）－３×１０￣（－４）ｍｏｌ／Ｌ）在大多数细胞可引起明显的膜去极化反应（５６／６０）。少数细胞对ＳＰ无反应（４／６０）。在ＳＰ去极化期间膜电导值有所增加,从平均值２．７２×１０￣（－８）ｍｈｏ增加２４．６％（ｎ＝３）。所测逆转电位值在＋４０－＋５０ｍＶ之间（ｎ＝３）。浊流平衡液（ＢＳＳ）中ＮａＣｌ以氯化胆碱置代,或用含ＴＴＸ（１０￣（－５）ｍｏｌ／Ｌ）的ＢＳＳ灌流,可使ＳＰ－去极化幅值大大减小但不能完全消除。而高（２０ｍｍｏｌ／Ｌ）和低（０ｍｍｏｌ／Ｌ）Ｃａ￣（２＋）的ＢＳＳ灌流时,使ＳＰ－去极化幅值相应的增加和降低。用含１０￣（－４）ｍｏｌ／ＬＣｄ￣（２＋）及１０￣（－２）ｍｏｌ／ＬＴＥＡ的ＢＳＳ灌流,均使ＳＰ－去极化明显减小。     

P2X_3受体在感觉神经节的表达

用免疫组织化学与原位杂交研究 P2 X3受体在背根神经节、三叉神经节和结状神经节的分布。结果显示 :1.原位杂交 :在三种感觉神经节中 ,95 %左右的神经节细胞为 P2 X3m RNA阳性 ,中、小型神经节细胞的杂交信号一般要比大型的神经节细胞强一些。 2 .免疫组织化学 :免疫组织化学结果与原位杂交结果基本一致。此外 ,在各神经节内 ,均显示出许多P2 X3免疫阳性神经纤维 ,在足掌表皮也显示许多 P2 X3免疫反应阳性纤维。结果提示 :P2 X3不仅参与机体的痛觉的形成 ,还可能参与其它感觉 ,如本体感觉等的形成     

P2X7受体在病理性疼痛中的研究进展

病理性疼痛主要包括组织损伤或炎症引起的炎症痛、神经系统损伤或疾病引起的神经病理性疼痛和恶性肿瘤及治疗引起的癌症痛三大类。病理性疼痛对常规的镇痛药物反应不理想,迫切需要寻找新的对病理性疼痛更有效和更特异的治疗手段。P2X7受体作为离子通道型嘌呤能受体,在炎症痛、神经病理性疼痛和癌症痛中都具有重要作用。靶向P2X7受体的新药物将为病理性疼痛的治疗带来新的希望。该文综述了P2X7受体在三类病理性疼痛中的研究进展。     

包含P2X3亚基的受体在介导痛觉中的作用

包含P2X3亚基的受体为三磷酸腺苷 (ATP)门控的阳离子通道 ,包括P2X3亚基的同源多聚体(P2X3受体 )和异源多聚体 (P2X2 /3受体 )。大量研究表明包含P2X3亚基的受体在介导多种类型痛觉中有重要作用     

福尔马林致痛对大鼠脊髓和背根神经节的P2X3的影响

目的:探索福尔马林致痛后大鼠脊髓和背根神经节(dorsal root ganglion,DRG)的P2X3表达变化。方法:选取健康成年正常SD大鼠25只,分正常对照组和实验组;实验组为右侧足底皮下给予0.1ml 5%福尔马林,分别观察15min、30min、1h、3h后处死,采用免疫组织化学方法及图像分析技术检测脊髓腰段及L4～6背根节P2X3的表达情况。结果:与正常对照组相比,实验15min、30min、1h组脊髓后角Ⅱ层P2X3表达未见变化,实验3h组可见P2X3表达升高,但未见明显差异;实验15min、30min组DRG神经元P2X3表达未见变化,1h组开始表达上调,3h组表达明显升高,与各组相比有显著性差异。结论:福尔马林致痛能引起脊髓和背根神经节P2X3的表达上调,可能是其产生伤害性作用的机制之一。     

P2X7受体敲除对小鼠骨癌痛的影响北大核心CSCD

癌痛是临床晚期恶性肿瘤患者常见的临床表现之一。其中,以肺癌、乳腺癌和前列腺癌等骨转移引起疼痛尤为严重。P2X7受体是ATP门控离子通道型嘌呤能受体的一个亚型,在脊髓背角主要表达在胶质细胞。P2X7受体激活可以促进胶质细胞释放多种炎症介质,介导脊髓中枢敏化。该受体在炎症痛及神经病理性疼痛中的作用已多有报道,但在癌痛中的作用尚有争议。本研究采用C57BL/6J小鼠股骨骨髓腔内接种Lewis肺癌细胞所诱导的骨癌痛小鼠模型,分析对比了野生型小鼠和P2X7受体基因敲除(P2rx7-/-)小鼠骨癌痛的发生、发展。野生型C57BL/6J小鼠股骨骨髓腔内接种Lewis肺癌细胞后,患侧后肢分别在第7和14天开始出现明显的触诱发痛和热痛过敏,并呈进行性加重;Cat Walk步态分析显示骨癌第21和28天,小鼠患侧脚印面积明显减小,站立时相持续时间缩短,举步时相持续时间显著延长;组织病理学结果显示受累骨骨髓腔有大量肿瘤细胞浸润,骨髓质正常结构消失,伴有髓质骨和皮质骨的破坏。与研究设计时的预期相反,P2rx7-/-小鼠接种瘤细胞后,患肢痛行为检测结果与野生型小鼠相似,甚至在Cat Walk步态分析检测值变化发生的时间上较野生小鼠有所提前。这与本研究组前期在大鼠骨癌痛模型观察到的阻断P2X7受体明显对抗骨癌痛的结果完全不同,提示P2X7受体在大、小鼠骨癌痛中可能发挥不同的作用,并再次提示疾病动物模型上的研究结果与人类疾病机理之间还存在巨大差异。     

克隆的P2受体亚型的药理学研究进展

细胞外嘌呤（腺苷,ADP,ATP）及嘧啶（UDP,UTP）为重要的信使分子,通过细胞表面P2受体介导产生不同的生物效应,P2嘌吟受体的概念于1978年被提出,随后根据药理学特征又被分为P2X及P2X嘌呤受体,90年代,采用分子生物学手段,一系列配体门控的P2X受体及G蛋白耦联的P2Y受体被克隆及功能表达,迄今为止,已有七型P2X受体亚型（P2X1－7）及六型P2Y受体亚型被克隆（P2Y1,2,4,6,11,12）,各型具有不同的分子结构,药理学特征及组织分布,本文还讨论了目前可用于区分各亚型激动剂及拮抗剂。     

Analgesic effect of electroacupuncture on bone cancer pain in rat model: the role of peripheral P2X3 receptor

Upregulation of P2X3 receptor (P2X3R) has been strongly implicated in nociceptive signaling including bone cancer pain (BCP). The present study, using rat bone cancer model, aimed to explore the role of P2X3R in regulating rat pain behavior under the intervention of electroacupuncture (EA). The BCP model was successfully established by injection with MRMT-1 breast cancer cell into the medullary cavity of left tibia for 3 × 10⁴ cells/3 μL PBS in rats as revealed by obvious bone destruction, decreased paw withdrawal thresholds (PWTs), and reduced paw withdrawal latencies (PWLs). Western blot analyses showed that P2X3R expression was significantly upregulated in ipsilateral lumbar 4–6 (L4-6) dorsal root ganglia (DRG), but the difference not seen in spinal cord dorsal horn (SCDH). With the in-depth study of P2X3R activation, we observed that intrathecal injection of P2X3R agonist α,β-meATP aggravated MRMT-1 induced BCP, while injection of P2X3R inhibitor A-317491 alleviated pain. Subsequently, we demonstrated that BCP induced mechanical allodynia and thermal hyperalgesia were attenuated after EA treatment. Under EA treatment, total P2X3R protein expression in ipsilateral DRGs was decreased, and it is worth mentioning that decreased expression of P2X3R membrane protein, which indicated that both the expression and membrane trafficking of P2X3R were inhibited by EA. The immunofluorescence assay showed that EA stimulation exerted functions by reducing the expression of P2X3R-positive cells in ipsilateral DRGs of BCP rats. Ca²⁺ imaging analysis revealed that the EA stimulation decreased the percentage of α,β-meATP responsive neurons in DRGs and inhibited calcium influx. Notably, the inhibitory effect of EA on mechanical allodynia and nociceptive flinches was abolished by intrathecal injection of α,β-meATP. These findings demonstrated EA stimulation ameliorated mechanical allodynia and thermal hyperalgesia in rat model of MRMT-1-induced BCP. EA exerts analgesic effect on BCP by reducing the overexpression and functional activity of P2X3R in ipsilateral DRGs of BCP rats. Our work first demonstrates the critical and overall role of P2X3R in EA’s analgesia against peripheral sensitization of MRMT-1-induced BCP and further supports EA as a potential therapeutic option for cancer pain in clinic.     

Electroacupuncture alleviates diabetic neuropathic pain in rats by suppressing P2X3 receptor expression in dorsal root ganglia

Diabetic neuropathic pain (DNP) is a troublesome diabetes complication all over the world. P2X3 receptor (P2X3R), a purinergic receptor from dorsal root ganglion (DRG), has important roles in neuropathic pain pathology and nociceptive sensations. Here, we investigated the involvement of DRG P2X3R and the effect of 2 Hz electroacupuncture (EA) on DNP. We monitored the rats’ body weight, fasting blood glucose level, paw withdrawal thresholds, and paw withdrawal latency, and evaluated P2X3R expression in DRG. We found that P2X3R expression is upregulated on DNP, while 2 Hz EA is analgesic against DNP and suppresses P2X3R expression in DRG. To evaluate P2X3R involvement in pain modulation, we then treated the animals with A317491, a P2X3R specific antagonist, or α β-me ATP, a P2X3R agonist. We found that A317491 alleviates hyperalgesia, while α β-me ATP blocks EA’s analgesic effects. Our findings indicated that 2 Hz EA alleviates DNP, possibly by suppressing P2X3R upregulation in DRG.

     

Dorsal root ganglia P2X4 and P2X7 receptors contribute to diabetes-induced hyperalgesia and the downregulation of electroacupuncture on P2X4 and P2X7

Diabetic neuropathic pain (DNP) is highly common in diabetes patients. P2X receptors play critical roles in pain sensitization. We previously showed that elevated P2X3 expression in dorsal root ganglion (DRG) contributes to DNP. However, the role of other P2X receptors in DNP is unclear. Here, we established the DNP model using a single high-dose streptozotocin (STZ) injection and investigated the expression of P2X genes in the DRG. Our data revealed elevated P2X2, P2X4, and P2X7 mRNA levels in DRG of DNP rats. The protein levels of P2X4 and P2X7 in DNP rats increased, but the P2X2 did not change significantly. To study the role of P2X4 and P2X7 in diabetes-induced hyperalgesia, we treated the DNP rats with TNP-ATP (2’,3’-O-(2,4,6-trinitrophenyl)-adenosine 5’-triphosphate), a nonspecific P2X1–7 antagonist, and found that TNP-ATP alleviated thermal hyperalgesia in DNP rats. 2 Hz electroacupuncture is analgesic against DNP and could downregulate P2X4 and P2X7 expression in DRG. Our findings indicate that P2X4 and P2X7 in L4–L6 DRGs contribute to diabetes-induced hyperalgesia, and that EA reduces thermal hyperalgesia and the expression of P2X4 and P2X7.     

神经生长因子受体Trka在糖尿病大鼠膀胱和腰骶背根神经节的表达

目的研究糖尿病大鼠膀胱组织及背根神经节中神经生长因子受体Trka的表达变化及意义。方法建立糖尿病大鼠模型20只,以15只正常大鼠为对照,应用免疫组化方法,分别检测大鼠膀胱组织及背根神经节中Trka的表达情况。结果无论是在膀胱组织还是在背根神经节中,糖尿病组Trka的表达均低于对照组,差异有统计学意义（P〈0.01）。结论神经生长因子受体Trka的表达降低与糖尿病膀胱病变的发生有关。     

人白血病细胞系P2X7受体的表达及其介导的细胞内钙浓度变化

采用半定量RT-PCR和流式细胞术,在基因和蛋白水平研究了白血病细胞系U937、HL60和Ramos细胞P2X7受体的表达。荧光染料Fura-2／AM负载后,用荧光分光光度计测定P2X7受体激动剂三磷酸腺苷(adenosine 5′-triphosphate,ATP)和苯甲酰苯甲酸ATP(2′,3′-O-(4-benzoyl)benzoyl-ATP,BzATP)刺激前后细胞内钙离子浓度的变化,以确认其功能。结果表明：U937和HL60细胞系表达P2X7受体的mRNA和蛋白,Ramos不表达;在激动剂的刺激下,可引发U937和HL60细胞胞内钙浓度的显著升高,但对Ramos没有作用。当去除胞外钙离子时,ATP和BzATP刺激均不能引起U937和HL60细胞胞内钙离子浓度的升高。提示U937和HL60细胞表达P2X7受体的基因和功能蛋白,Ramos细胞则不表达该受体。     

Prenatal expression of purinergic receptor P2X3 in human dorsal root ganglion

The dorsal root ganglion (DRG) is consisted of neurons that relay multiple types of spinal sensory stimuli to the central nervous system. Several neuroactive molecules may be involved in sensory modulation especially pain processing at the DRG, including the purinergic receptor P2X3 and calcitonin-gene-related peptide (CGRP). P2X3 receptor has been considered a promising pharmaceutical target for the development of new pain medicine. Currently, litter is known about the expression of P2X3 in the human DRG. The present study characterized the localization of P2X3 in prenatal human DRG obtained from fetuses at 4-8 gestational months, by comparing to CGRP expression as well as binding pattern of isolectin-B4 (IB4), a marker of small DRG neurons presumably relevant to nociception. P2X3 immunoreactivity (IR) appeared in most neuron-like perikarya, with their numerical density reduced during the gestational period studied. P2X3 IR was co-labeled very commonly with IB4 binding and infrequently with CGRP IR and was not colocalized with IR for the gliocyte marker glutamine synthetase. Together, the data show an early and broad expression of P2X3 in prenatal human DRG neurons, pointing to a biological role of purinergic signaling during the development of spinal sensory system.