CN109536530A - 一种利用trpv1启动子及光遗传学手段特异性抑制疼痛生成与传递的方法 - Google Patents
一种利用trpv1启动子及光遗传学手段特异性抑制疼痛生成与传递的方法 Download PDFInfo
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Abstract
本发明涉及到一种利用TRPV1启动子及光遗传学手段在特异性地抑制体内疼痛神经元兴奋进而抑制疼痛生成与传递的方法，是一种新的非侵入性、特异性缓解疼痛的方法。用TRPV1启动子驱动光抑制性敏感蛋白ArchT特异性的表达在动物DRG的中小型神经元即伤害性感受器中，当给予532nm的绿色激光刺激时，ArchT蛋白会将神经细胞中的H+泵到细胞外使细胞超极化而被抑制，从而达到抑制疼痛的效果。这是一种全新的、非侵入性的、特异性的抑制疼痛的方法。该研究势必会为以后镇痛的基因治疗奠定良好的理论与实践基础。
Description
技术领域
本发明涉及到一种利用TRPV1启动子及光遗传学手段在特异性地抑制体内疼痛神经元兴奋进而抑制疼痛生成与传递的方法。广泛地应用于生物学、光遗传学、神经科学领域，具体地涉及用TRPV1启动子、光抑制性敏感蛋白ArchT和AAV5构建了一种重组AAV病毒(AAV5-TRPV1-ArchT-eGFP)，将其直接注射到动物的DRG中，待目的蛋白ArchT高量并稳定表达在神经元细胞膜后研究其对神经***及动物疼痛反应的抑制作用
背景技术
慢性疼痛是一种病理性状态，大大降低人们的生活质量。然而迄今为止，仍然没有特异性治疗慢性疼痛的方法。目前可使用的镇痛药物既缺乏针对性同时又具有严重的副作用。因此建立一种非侵入性、特异性抑制疼痛的方法就变得非常重要。
近年来研究发现，因为疼痛就医的病人占病人总量的40%以上，其中又有20%的疼痛病人遭受着超过六个月以上的慢性疼痛的折磨。慢性疼痛给病人带来极大的痛苦，大大降低人们的生活质量。很多病人因为慢性疼痛的折磨无法正常睡眠与工作，更严重的会导致由慢性疼痛转变为部分或者完全残疾。然而目前对于疼痛的治疗主要是通过药物、外科手术、电刺激和躯体康复治疗等技术手段。通过外科手术的方法治疗疼痛，除了给病人带来比较大的外部创伤外，伤口也比较难恢复，同时手术费用昂贵。电刺激、针灸、推拿等物理疗法也有很大的弊端，如镇痛效果不显著、治疗周期长等。而临床上最常用的方法则是药物治疗。阿片类受体激动剂***、谷氨酸受体拮抗剂NS1209和***、Na+通道阻断剂美西律和拉莫三嗪、K+通道开放剂瑞替加滨和氟吡汀以及GABA调节剂安定等常常作为镇痛药物使用。这些药物既缺乏针对性同时又具有严重的副作用。例如***的使用可造成剂量依赖性的增加，临床表现为药物成瘾，使患者承受很大的痛苦。
目前光遗传学 (optogenetics)在神经科学领域应用十分广泛。所谓光遗传学是指，把光学与遗传学的手段结合起来，用以激活或者失活特定组织及细胞的学问与技术。到目前为止已有大量光遗传学蛋白被表达并应用在不同生物的神经***，其中抑制性光敏感蛋白有：Halorhodopsin (NpHR) 、Archaerhodopsin-3和ArchT等。近年来研究表明相比于NpHR，Arch可以快速恢复由光诱导的失活状态，降低光敏感蛋白对光的不应期。ArchT不仅具有Arch同样的功能，且光敏感性比Arch高三倍以上。ArchT可以稳定并高量的表达在神经细胞膜上，同时也可以很好的表达在神经突起上。体外光照情况下ArchT可以介导产生最大约900pA的光电流使细胞膜超级化，从而介导对神经***的抑制作用。
光遗传学的主要方法是利用不同类型的病毒载体，把光敏感蛋白引入离体或在体动物神经***中。然后根据不同的光敏感蛋白使用不同波长的光照射，激活该光敏感蛋白使其发挥作用达到激活或者失活特定神经细胞的目的。目前由于腺相关病毒（AAV）具有自然缺陷、安全性好、无致病源性、宿主细胞范围广、在体内表达外源基因时间长等特点，被广泛应用与这种病毒载体，携带光敏感蛋白进入并表达在宿主神经细胞中。AAV有十几种血清型，包括AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10等。研究表明直接DRG注射的情况下AAV5具有最高的感染效率。
目前对于疼痛的治疗没有特异性，本发明用一种特异性的启动子驱动光抑制性敏感蛋白特异表达在背根神经节 (DRG)的伤害性感受器 (nociceptor)中，达到特异性抑制疼痛的目的。辣椒素受体TRPV1是一种非选择性的阳离子通道，稳定并高量表达在DRG的伤害性感受器中。本发明利用TRPV1启动子的特异性将ArchT特异地表达在伤害性感受器(nociceptor)中，从而建立起一种新的非侵入性、特异性和快速可控地抑制疼痛传递的镇痛方法。本发明可以阐释光作为一种新的光遗传学策略在治疗疼痛方面的应用。
发明内容
使用直接DRG注射的方法仅仅将目的蛋白ArchT表达在DRG的中小型神经元中，也就是只涉及外周神经***的微小改变，中枢神经***被完全排除在外。这种方法在达到治疗疼痛的同时尽量不损失外周神经***，更不会损害中枢神经***。也就是在抑制外周神经***特定细胞活性的同时不会影响中枢神经***的正常功能。
通过TRPV1启动子特异性的将光抑制性敏感蛋白ArchT表达在DRG的伤害性感受器中，这些阳性细胞并不涉及控制触觉和痒的神经元。本发明抑制疼痛的同时并不会影响触觉和痒信息的正常传递和感知。本发明不涉及中枢神经***的加工，而是直接从外周抑制DRG中伤害性感受器的活性，即切断其向中枢神经***的传递。本发明没有疼痛类型的限制，因此不仅可以治疗不同类型的疼痛，并且可以同时治疗多种类型的疼痛。
附图说明
图1为重组TRPV1载体
图2为ArchT与TRPV1在DRG中的共定位
图3为光遗传学抑制ArchT+神经元可以缓解小鼠的机械痛和热痛，其中：
A.功能性抑制ArchT+神经元的活性但仍保持其发育完整性的实验原理图；
B. ArchT介导的机械痛和热痛行为学实验原理图；
C.在绿色光照和无光照情况下的ArchT+小鼠 (n=10)与WT未注射病毒小鼠 (n=9)的机械缩足反应阈值；
D.在绿色光照和无光照情况下的ArchT+小鼠 (n=5)与WT未注射病毒小鼠 (n=5)的热辐射缩足反应阈值。
具体实施方式
1. PCR反应体系及产物的纯化
引入酶切位点SnaBI和XhoI扩增TRPV1启动子目的片段。
上游引物SnaBI TRPV1F: GCATACGTAGAGGACCAGAAGAAGGAGAGTC
下游引物XhoI TRPV1R: GTACTCGAGGTGCAGGCACACTCCAAATG
PCR反应体系：
PCR循环体系：
2. 双酶切反应体系及产物的纯化
2.1将胶回收纯化的TRPV1启动子目的片段与pEGFP-N1载体双酶切，30℃水浴酶切1小时。
双酶切反应体系：
2.2 双酶切产物的纯化
双酶切产物经琼脂糖凝胶电泳后割胶，用Axygene胶回收试剂盒回收纯化。
3. 利用TRPV1启动子将目的基因表达在离体DRG神经元中
将TRPV1启动子克隆到pEGFP-N1载体中取代该载体本身的CMV启动子。该产物在本发明中被命名为TRPV1载体。利用脂质体转染的方法将TRPV1载体转染到体外培养的DRG神经元中。
3.1克隆TRPV1载体
TRPV1启动子是TRPV1基因转录起始位点前约2kb的DNA片段，从NCBI搜索其基因序列为2069bp (Gene: Trpv1 ENSMUSG00000005952)，并引入SnaBI和XhoI两个酶切位点设计引物，方法是从C57BL/6小鼠基因组中经PCR获得TRPV1启动子序列。
PCR所得的DNA片段长约2kb，可确定为TRPV1启动子序列。将TRPV1启动子重组的载体命名为TRPV1载体 (图1) 。
图1 重组TRPV1载体
DRG的中小型神经元负责伤害性刺激引起的痛觉信息的传递，而DRG的大细胞则负责非伤害性刺激引起的触觉信息的传导。ArchT在DRG中的特异性表达说明：当ArchT被特定波长范围内的光照射激活后，可以特异性的抑制疼痛，同时又不会影响机体对触觉信息的正常传递与感知。
3.2 ArchT与TRPV1共定位
构建重组AAV病毒用的是TRPV1启动子。用TRPV1抗体做了重组AAV病毒注射两个月后的小鼠DRG切片染色，观察ArchT是否与TRPV1在DRG中共定位。
图2 ArchT与TRPV1在DRG中的共定位
免疫染色结果显示，ArchT与TRPV1在DRG中几乎完全重叠 (图2)，证明了TRPV1启动子的高度特异性。同时由于ArchT与TRPV1在DRG中的高度共定位， ArchT是高表达在TRPV1+神经元中的，那么当给以532nm的绿色激光照射时，由于ArchT蛋白的激活，ArchT发挥其光敏感性抑制作用就会将TRPV1+神经元抑制。而TRPV1是一种非选择性的阳离子通道，可以被伤害性的化学刺激：辣椒素、质子 (pH < 5.9)和伤害性热刺激 (>43 °C)所激活，所以TRPV1与热刺激有着密切的关联。当ArchT发挥其光敏感性抑制作用将TRPV1+神经元抑制的时候，热痛行为将同时受到严重的影响，会在很大程度上抑制热痛。
4.光抑制ArchT+ 神经元后可缓解机械痛和热痛
将重组AAV病毒注射两个月后并成功使ArchT特异性表达在DRG的伤害性感受器中的小鼠用来做疼痛相关的行为学测试。此测试可用来评估光照对ArchT阳性神经元的抑制效果。图3 A展示了功能性抑制ArchT阳性神经元的实验原理图。在功能性抑制ArchT+的过程中，DRG中的伤害性感受器仅仅被功能性抑制，其生长发育等其他生理行为都可继续完好的进行。重组AAV病毒注射后的小鼠被用来做机械痛和热痛行为学测试，分别通过使用机械刺激和热辐射刺激在532nm绿色激光和无光照情况下照射小鼠的后脚掌 (图3 B)。在对照组WT小鼠中，532nm绿色激光 (机械痛行为实验使用的光强为：1.0-1.5mW/mm2，热痛行为实验使用的光强为：0.15-0.2 mW/mm2)并没有诱导小鼠的机械缩足反应阈值（图3 C）和热辐射缩足反应阈值 (图3 D)产生异常。然而，用同样光照强度的532nm绿色激光照射ArchT实验组小鼠，其机械缩足反应阈值 (图3 C)和热辐射缩足反应阈值 (图3 D)相对于无光照情况下都有显著性的增加。光抑制DRG的伤害性感受器中ArchT+神经元的活性可以有效缓解小鼠的机械痛和热痛。
图3 光遗传学抑制ArchT+神经元可以缓解小鼠的机械痛和热痛，其中：
A.功能性抑制ArchT+神经元的活性但仍保持其发育完整性的实验原理图；
B. ArchT介导的机械痛和热痛行为学实验原理图；
C.在绿色光照和无光照情况下的ArchT+小鼠 (n=10)与WT未注射病毒小鼠 (n=9)的机械缩足反应阈值；
D.在绿色光照和无光照情况下的ArchT+小鼠 (n=5)与WT未注射病毒小鼠 (n=5)的热辐射缩足反应阈值。数据代表means ± S.E.M，分析使用Student’s T-test. **P < 0.01,***P < 0.001.
本发明方法可以在动物成年，一切生理及心理功能成熟后，通过直接将光敏感性抑制蛋白表达在特定细胞群中，光照后达到对该细胞群功能的抑制，来研究该细胞群被抑制后实验组会产生什么样的表型变化。因此当我们想研究某种类型细胞群的功能时，就可以选择该细胞群特异性的启动子，驱动光抑制性敏感蛋白ArchT特异的表达在该细胞群中，通过光照抑制该细胞群的活性，研究该细胞群在机体中扮演的角色。其结果是：该细胞群仍然完好的存在于机体中，没有丝毫损伤，只是其细胞活性被抑制，同时该细胞群与周围其他类型的细胞之间的物质及能量传递等相互作用依然可以很好的进行。这种方法没有发育代偿，同时由于ArchT对光照刺激反应快速高效，因此是一种快速可控的研究方法。利用快速的、可控的光遗传学工具建立了一种新的非侵入性和特异性抑制疼痛传递的镇痛方法，光作为一种新的光遗传学策略在治疗疼痛方面应用。
Claims (4)
1.一种利用TRPV1启动子及光遗传学手段特异性地抑制体内疼痛神经元兴奋进而抑制疼痛生成与传递的方法。
2.根据权利要求1所述的一种利用TRPV1启动子及光遗传学手段在特异性地抑制体内疼痛神经元兴奋进而抑制疼痛生成与传递的方法，把光敏感蛋白引入离体和在体动物神经***中，根据不同的光敏感蛋白使用不同波长的光照射，激活该光敏感蛋白使其发挥作用达到激活或者失活特定神经细胞的方法。
3.根据权利要求2所述的一种利用TRPV1启动子及光遗传学手段在特异性地抑制体内疼痛神经元兴奋进而抑制疼痛生成与传递的方法，利用TRPV1启动子将目的基因表达在离体和在体DRG神经元中。
4.根据权利要求2所述的一种利用TRPV1启动子及光遗传学手段在特异性地抑制体内疼痛神经元兴奋进而抑制疼痛生成与传递的方法，ArchT高量并长期稳定地表达在体内DRG神经元中。
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