CN114712511A - Hap1在筛选神经病理性疼痛治疗药物中的应用 - Google Patents
Hap1在筛选神经病理性疼痛治疗药物中的应用 Download PDFInfo
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Abstract
本发明公开了HAP1在筛选神经病理性疼痛治疗药物中的应用，属于生物医药技术领域。本发明选用野生型老鼠作为对照组、HAP1杂合子小鼠作为低表达实验组，依次构建神经病理急性疼痛和慢性疼痛模型，分别检测了两组小鼠的术后疼痛反应。结果发现，降低HAP1的表达水平，可以显著缓解病理性疼痛反应。这一发现可以为日后的疼痛研究提供新的分子靶点，也可以为临床疼痛治疗提供新的理论支持。
Description
技术领域
本发明属于生物医药技术领域，具体涉及HAP1在筛选神经病理性疼痛治疗药物中的应用。
背景技术
神经病理性疼痛（Neuropathic Pain, NP）是一种持久的且伤害性大的慢性疼痛，国际疼痛研究协会定义为：由神经系统损伤或疾病所引起的疼痛，特点是无法解释的广泛性疼痛、感觉障碍、伴随灼烧感、痛觉过敏或者在缺乏伤害性刺激情况下出现了疼痛，即为痛觉过敏、超敏和自发性疼痛，是公认的治疗难度大的慢性疼痛之一。流行病学统计发现约6.9%-10%的慢性疼痛患者具有神经病理性疼痛症状。由于 NP 强烈且持久，常常可抵抗所有可用的镇痛措施，对患者而言不仅是身体的摧残，更是心理的折磨，极大降低了患者的生活质量。同时 NP 病因复杂，有关其具体的发生发展机制尚不十分清楚。因此，深入研究NP的分子机制对于寻找新的镇痛靶点十分重要。
背根神经节（Dorsal root ganglion，DRG）主要负责传送伤害性刺激信息，而脊髓背角（Spinal dorsal horn, SDH）是接收和处理疼痛信息的初级门户。伤害性刺激信息在此经过初步整合后，一方面作用于前角运动细胞，引起局部的防御性反射，另一方面则继续向上传递至大脑中枢。脊髓通过“闸门控制”机制在局部参与伤害性信息传入的调节，是疼痛信号处理的初级调节中枢。亨廷顿蛋白相关蛋白1（Huntingtin associated protein 1，HAP1），是一种著名的神经蛋白，广泛表达在中枢和周围神经系统中，也在脊髓和背根神经节中表达较高。HAP1具有十分重要的作用，如帮助细胞内的囊泡转运，例如脑源性神经营养因子、14-3-3蛋白、鸟嘌呤核苷酸交换因子等；调节膜受体蛋白的循环和表达，包括γ-氨基丁酸A型受体、雄激素受体、L型钙通道、酪氨酸受体激酶A。免疫染色结果显示，70-80%的HAP1阳性神经元与伤害性或机械感受标记物共定位，如CGRP、SP、NOS、TRPV1等，表明HAP1可能对疼痛具有潜在的调节作用。
因此，本发明的主要目的是研究不同HAP1表达水平，是否可以影响神经病理性急性、慢性疼痛行为学的改变，从而进一步为深入研究疼痛相关机制提供理论基础。
发明内容
本发明的目的是提供HAP1在筛选神经病理性疼痛治疗药物中的应用。
在本发明中，选用野生型老鼠作为对照组、HAP1杂合子小鼠作为低表达实验组，依次构建神经病理急性疼痛和慢性疼痛模型，分别检测了两组小鼠的术后疼痛反应（机械刺激痛、热敏反应、冷敏反应）。结果发现，降低HAP1的表达水平，可以显著缓解病理性疼痛反应。这一发现可以为日后的疼痛研究提供新的分子靶点，也可为临床疼痛治疗提供新的理论支持。
附图说明
图1为实施例1中构建SNI 急性疼痛模型后检测机械刺激的阈值变化结果。
图2为实施例2中构建SNI 急性疼痛模型后检测热刺激的阈值变化结果。
图3为实施例3中构建SNI 急性疼痛模型后检测冷刺激的阈值变化结果。
图4为实施例4中构建CCI慢性疼痛模型后检测机械刺激的阈值变化结果。
图5为实施例5中构建CCI慢性疼痛模型后检测热刺激的阈值变化结果。
图6为实施例6中构建CCI慢性疼痛模型后检测冷刺激的阈值变化结果。
图1至图6中，HAP1+/+为对照组，HAP1+/-为实验组。
具体实施方式
神经病理性疼痛是一种持久且伤害性大的慢性疼痛，是公认的治疗难度很大的一种慢性疼痛。流行病学统计发现约6.9%-10%的慢性疼痛患者具有神经病理性疼痛症状。由于疼痛感强烈且持续时间长久，导致镇痛药物和治疗手段效果甚微。不仅摧残着患者的身体，更是心理的折磨。神经病理性疼痛病因复杂，广泛涉及多种信号通路和蛋白分子。因此，深入研究神经疼痛的蛋白作用，可以为寻找临床疼痛治疗提供新的理论基础。
HAP1是一种大量表达在中枢和外周神经系统的蛋白分子，具有调节神经元发育、囊泡分泌和受体运输等多个功能的蛋白质。研究报道HAP1在脊髓后角和神经节中高度表达，然而关于HAP1在脊髓中的作用及其是否具有调节疼痛的功能尚未明确。在本发明中选用了急性和慢性两种疼痛模型，分别检测HAP1低表达是否可以缓解疼痛行为。
下面结合附图和具体实施例对本发明作进一步详细说明，但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明的范围。实施例中未注明具体条件的实验方法及未说明配方的试剂均为按照本领域常规条件。
HAP1基因敲除小鼠购自于美国Jackson Laboratory (Strain #007749; BarHarbor, Maine, USA)。C57BL/6来源的HAP1杂合子进行繁殖，可获得三种基因型小鼠，分别是：纯合子（HAP1-/-，homozygous, KO），杂合子（HAP1+/-，heterozygous, HET），野生子（HAP1+/+，wild type, WT），并采用PCR技术进行基因型鉴定，引物如下：
WT小鼠序列：5’-ttttggaggtctggtctcgctctg-3’ (SEQ ID NO.1)
5’-cgtcttccatcttagtgcgttcac-3’ (SEQ ID NO.2)
KO小鼠序列：5’-ttttggaggtctggtctcgctctg-3’ (SEQ ID NO.3)
5’-cttcatgtggatgctagggatcc-3’ (SEQ ID NO.4)。
下列实施例中，采用的实验方法为：
1、急性疼痛（Spared nerve injury，SNI）模型构建
启动小动物麻醉机，使用异氟烷进行全身麻醉。在左侧小腿后侧，依次剥离皮肤、肌肉，暴露坐骨神经（可见三个神经分支：胫神经、腓总神经、腓肠神经）。使用6.0丝线结扎胫神经和腓总神经，剪断并切除5 mm神经末端。手术过程务必小心，不可损伤和触碰腓肠神经，任何牵张或损伤都谨慎避免。依次缝合肌肉和皮肤。
2、慢性疼痛（Chronic constriction injury，CCI）模型
启动小动物麻醉机，使用异氟烷进行全身麻醉。在左侧小腿后侧，依次剥离皮肤、肌肉，暴露坐骨神经。在三叉神经周围小心结扎3次，每个绳结间隔1mm。小心打结6.0丝线，直至看到同侧后肢轻微摆动。依次缝合肌肉和皮肤。
3、Von Frey机械痛实验
测试前1h将小鼠放在金属网笼子里适应环境。使用一系列不同硬度(0.02-2.56gf；Stoelting)的von Frey细丝刺激小鼠后爪，垂直接触足底皮肤并记录反应。每个小鼠检测3次，取平均值。
4、Hargreaves辐射热实验
测试前1h将小鼠放在相应的笼子里适应环境。通过Hargreaves辐射热设备(IITCLife Science)评估热敏反应。设定小鼠术前热敏阈值为8-12秒，最长辐射时间为20秒，以防止组织受损。分别记录不同小鼠的缩足时间。每只老鼠检测3次，取平均值。
5、丙酮试验
选用丙酮检测冷刺激反应。向小鼠足底后爪中部喷射20μl丙酮。丙酮挥发会引发局部温度变低，以此检测对冷刺激的反应。小鼠行为可分为4级：0，无反应；1，快速缩爪、轻弹后爪；2，快速缩爪、长时间抬爪或反复摇晃；3，反复摇晃后爪、抬爪不触碰地面、持续舔爪。每只老鼠检测3次，取平均值。
实施例1
构建急性疼痛（Spared nerve injury，SNI）模型，检测机械刺激的阈值变化
分别选用野生型小鼠（WT，对照组）和HAP1杂合子小鼠（HET, 实验组），采用SNI坐骨神经分支切断手术，构建神经病理性疼痛急性模型。术后4天开始选用Von Frey方法检测不同组别小鼠机械刺激痛的阈值变化。
如图1所示，对照组小鼠表现出持续且强烈的疼痛反应，实验组小鼠亦有疼痛反应，但是疼痛反应显著低于对照组，这表明HAP1蛋白水平降低可以缓解急性疼痛。
实施例2
构建急性疼痛（Spared nerve injury，SNI）模型，检测热刺激的阈值变化
分别选用野生型小鼠（WT，对照组）和HAP1杂合子小鼠（HET, 实验组），采用SNI坐骨神经分支切断手术，构建神经病理性疼痛急性模型。术后4天开始选用热板实验检测不同组别小鼠热敏反应的阈值变化。
如图2所示，对照组小鼠表现出强烈的热敏反应，热光源照射后缩脚时间短；而实验组小鼠缩脚的时间显著长于对照组，说明HAP1表达降低可以缓解急性疼痛诱导的热敏反应。
实施例3
构建急性疼痛（Spared nerve injury，SNI）模型，检测冷刺激的阈值变化
选用野生型小鼠（WT，对照组）和HAP1杂合子小鼠（HET, 实验组），采用SNI坐骨神经分支切断手术，构建神经病理性疼痛急性模型。术后4天开始选用丙酮实验检测不同组别小鼠冷刺激反应的阈值变化。
如图3所示，对照组小鼠表现出强烈的冷刺激反应，接触到丙酮后快速缩脚，伴有舔足，甩足的剧烈反应；而实验组小鼠在术后初期也具有类似反应，但是后期逐渐表现出较弱的冷刺激反应，说明HAP1表达降低可以缓解急性疼痛后期的冷刺激反应。
实施例4
构建慢性疼痛（Chronic constriction injury，CCI）模型，检测机械刺激的阈值变化
选用野生型小鼠（WT，对照组）和HAP1杂合子小鼠（HET, 实验组），采用CCI坐骨神经结扎手术，构建神经病理性疼痛慢性模型。术后4天开始选用Von Frey方法检测不同组别小鼠机械刺激痛的阈值变化。
如图4所示，对照组小鼠表现出持续且强烈的疼痛反应，实验组小鼠的疼痛反应较弱；表明HAP1表达降低可以缓解慢性疼痛引发的机械性刺激痛。
实施例5
构建慢性疼痛（Chronic constriction injury，CCI）模型，检测热刺激的阈值变化
选用野生型小鼠（WT，对照组）和HAP1杂合子小鼠（HET, 实验组），采用CCI坐骨神经结扎手术，构建神经病理性疼痛慢性模型。术后4天开始选用热板实验检测不同组别小鼠热敏反应的阈值变化。
如图5所示，对照组小鼠对热源具有强烈的反应，热光源照射后快速缩脚并舔足；而实验组小鼠的热敏反应相对较弱，缩脚慢，说明HAP1表达降低可以缓解慢性疼痛诱导的热敏反应。
实施例6
构建慢性疼痛（Chronic constriction injury，CCI）模型，检测冷刺激的阈值变化
选用野生型小鼠（WT，对照组）和HAP1杂合子小鼠（HET, 实验组），采用CCI坐骨神经结扎手术，构建神经病理性疼痛慢性模型。术后4天开始选用丙酮实验检测不同组别小鼠冷刺激反应的阈值变化。
如图6所示，对照组小鼠表现出强烈的冷刺激反应，接触到丙酮后快速缩脚，伴有舔足或是甩足的剧烈反应；实验组小鼠的术后初期反应类似，但是后期逐渐表现出较弱的冷刺激反应，说明HAP1表达降低可以缓解慢性疼痛后期的冷刺激反应。
序列表
<110> 南通大学
<120> HAP1在筛选神经病理性疼痛治疗药物中的应用
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Claims (2)
1.HAP1在筛选神经病理性疼痛治疗药物中的应用。
2.根据权利要求1所述的应用，其特征在于：所述神经病理性疼痛为神经病理急性疼痛或神经病理慢性疼痛。
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| CN1382709A (zh) * | 2001-04-26 | 2002-12-04 | 上海博德基因开发有限公司 | 一种多肽——哚蛋白－50.16和编码这种多肽的多核苷酸 |
| US20100249065A1 (en) * | 2009-03-24 | 2010-09-30 | Nadine Cohen | Biomarkers for Assessing Peripheral Neuropathy Response to Cancer Treatment |
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| CN1382709A (zh) * | 2001-04-26 | 2002-12-04 | 上海博德基因开发有限公司 | 一种多肽——哚蛋白－50.16和编码这种多肽的多核苷酸 |
| US20100249065A1 (en) * | 2009-03-24 | 2010-09-30 | Nadine Cohen | Biomarkers for Assessing Peripheral Neuropathy Response to Cancer Treatment |
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