转录因子Tlx3在瘙痒和疼痛感受神经元发育中的功能研究

发布时间：2018-04-24 02:22

本文选题：瘙痒 + 疼痛　；参考：《中国人民解放军军事医学科学院》2017年博士论文

【摘要】：瘙痒和疼痛是机体十分重要的两种感觉,在物种的进化过程中比较保守。在生理条件下,短暂的瘙痒和疼痛是一种保护机制,它能提示机体外界的有害刺激,从而做出相应的反应来避免进一步的伤害,比如疼痛主要是引起一种退缩反应,而瘙痒则引起强烈的抓挠欲望。虽然二者在个人体验以及反应形式上差异巨大,但是其并不是完全独立的,二者之间存在紧密的联系。一些和疼痛相关的离子通道和受体也介导瘙痒信息的传递。随着瘙痒研究的逐步深入,瘙痒相关受体和通路逐渐被发现,这些受体参与的瘙痒信息的传递也需要部分疼痛相关离子通道的参与,比如TRPA1和TRPV1。疼痛和瘙痒之间还存在抑制关系,越来越多的证据表明疼痛确实能够抑制瘙痒,而且一些相关的抑制通路也陆续被报道。相比于对疼痛的研究,瘙痒的研究本世纪以来才有了比较大的进展,虽然部分瘙痒相关的受体、离子通道、神经递质以及神经元陆续被发现,但是关于这些分子的表达以及相关神经元发育的调控机制,目前来说还很不清楚。一些文献报道,部分转录因子参与感觉神经元的发育调控,我们实验室前期工作表明转录因子Tlx3能够调控背根神经节中Trk A+神经元向Ret+神经元的分化,同时也能够控制一系列疼痛相关分子的表达。由于瘙痒感受神经元也是来源于Trk A+神经元,所以我们猜测Tlx3也能够调控瘙痒感受神经元的分化发育以及瘙痒相关分子的表达。为了探究这一问题,我们构建了Tlx3条件性敲除小鼠Tlx3F/F;Nav1.8-cre,即选择性地在背根神经节Nav1.8+神经元中敲除Tlx3。为了探究转录因子Tlx3是否会调控瘙痒相关分子的表达水平,我们首先用RNAseq的方法比较了Tlx3条件性敲除小鼠和对照组小鼠背根神经节的转录组差异,发现在Tlx3条件性敲除小鼠的背根神经节中大部分目前已经报道的瘙痒相关分子的表达明显下降。为了进一步验证RNAseq的结果,我们采用了m RNA原位杂交实验检测了部分瘙痒相关分子的表达,发现在Tlx3条件性敲除小鼠的背根神经节中Mrgpr A3,IL31ra,Nppb,Sst的表达完全缺失。以上结果证明了Tlx3是许多瘙痒相关分子的表达所必须的。为了探究Tlx3条件性敲除小鼠背根神经节中瘙痒相关分子的表达下调是否会影响小鼠的瘙痒行为,我们首先比较了Tlx3条件性敲除小鼠和对照组小鼠对多种致痒剂诱导产生的急性瘙痒的反应。我们选用了四种不同类型的致痒剂:Compound48/80,chloroquine,α-Me-5-HT,SLIGRL-NH2。Compound48/80能够引起组胺依赖性瘙痒,而后三种引起非组胺依赖性瘙痒。Tlx3条件性敲除小鼠对这四种致痒剂引起的急性瘙痒的反应和对照组小鼠相比都明显减弱,说明Tlx3能够调控急性瘙痒的产生,既包括组胺依赖性瘙痒也包括非组胺依赖性瘙痒。由于许多瘙痒相关受体也介导慢性瘙痒,所以我们又检测了Tlx3条件性敲除小鼠对慢性瘙痒的反应。我们采用了两种慢性瘙痒模型:皮肤干燥(Dry skin)和过敏性接触性皮炎(Allergic contact dermatitis,ACD)模型。Tlx3条件性敲除小鼠对于Dry skin引起的慢性瘙痒的反应比对照组小鼠弱,但是对于ACD引起的慢性瘙痒的反应却和对照组小鼠没有明显区别。说明Tlx3也参与调控部分类型慢性瘙痒的产生。由于许多瘙痒相关分子的表达都明显缺陷,但是Tlx3条件性敲除小鼠仍然出现了明显的瘙痒,提示慢性瘙痒的复杂性以及其他信号通路的参与。之前的报道表明在部分慢性瘙痒模型中TRPV1的表达出现上调,这提示其可能参与慢性瘙痒。我们发现在Tlx3条件性敲除小鼠和对照组小鼠中TRPV1拮抗剂AMG9810能明显缓解Dry skin以及ACD引起的慢性瘙痒,表明TRPV1确实参与慢性瘙痒。疼痛能够抑制瘙痒,而有报道表明背根神经节中的TRPV1+/TRPA1+神经元参与这种抑制,为了探究其是否也受Tlx3调控,我们使用了辣椒素(capsaicin)和AITC(allyl isothiocyanate)面部注射实验。我们发现面部注射AITC在Tlx3条件性敲除小鼠和对照组小鼠都只引起了明显的疼痛反应,而几乎没有瘙痒反应,说明抑制通路没有受到明显影响。但是面部注射辣椒素在Tlx3条件性敲除小鼠却引起了明显增强的疼痛反应,更意外的是同时也出现了明显增强的瘙痒。考虑到抑制通路是正常的,我们认为这种明显增强的瘙痒是由于辣椒素诱导的瘙痒本身增强,并且不能完全被抑制通路抑制才表现出来的。由于辣椒素在Tlx3条件性敲除小鼠内引起了明显增强的疼痛和瘙痒,所以我们检测了辣椒素的受体TRPV1的表达情况,发现其表达量在Tlx3条件性敲除小鼠的背根神经节,脊髓以及皮肤中都明显增加。为了详细地了解Tlx3条件性敲除小鼠背根神经节中TRPV1的表达模式,我们进行一系列的免疫荧光实验。我们发现在野生型小鼠的背根神经节中只有约27%Tlx3+神经元表达TRPV1而且绝大多数TRPV1+神经元都不表达或者低表达Tlx3,进一步证实Tlx3能够抑制TRPV1的表达。Tlx3条件性敲除小鼠的背根神经节中TRPV1大幅度地扩展到IB4+非肽类神经元中,同时其在CGRP+肽类神经元的比例也有增加。相似地,在Tlx3条件性敲除小鼠的脊髓背角,TRPV1+神经纤维的投射区域也扩展到IB4+神经纤维投射区域即二层内侧(lamina IIi)。考虑到Tlx3能够调控许多疼痛相关受体,而且TRPV1也是感受热刺激的主要受体之一,所以我们进行了一系列疼痛行为学实验来探究Tlx3是否调控疼痛的产生。我们发现Tlx3条件性敲除小鼠对冷刺激以及炎症性疼痛的反应性明显下降,而对于机械,热刺激以及神经病理性疼痛的反应却保持正常。由于TRPV1是目前已知的感受热的主要受体,虽然其表达水平在Tlx3条件性敲除小鼠中明显升高,但是Tlx3条件性敲除小鼠对热刺激的反应却没有明显变化。为了进一步验证这一现象,我们采用了脊髓神经元激活实验来判断热刺激是否能够在Tlx3条件性敲除小鼠的脊髓背角激活更多的神经元,结果发现热刺激在Tlx3条件性敲除小鼠和对照组小鼠脊髓内激活的脊髓神经元数量没有明显差别,而辣椒素在Tlx3条件性敲除小鼠脊髓内激活的神经元数量却明显比对照组小鼠多,说明Tlx3条件性敲除小鼠内明显增加的TRPV1使得其对辣椒素的敏感性增加,却不影响其对热刺激的反应。综上,本研究证实了转录因子Tlx3能够调控大部分瘙痒相关分子的表达,其功能缺失导致瘙痒相关分子的表达下降或者消失,从而使小鼠对急性瘙痒和部分慢性瘙痒的反应明显减弱。Tlx3能够抑制TRPV1的表达,Tlx3条件性敲除小鼠内TRPV1的表达明显增加,这就导致面部注射辣椒素在Tlx3条件性敲除小鼠内引起了同时增强的疼痛和瘙痒。Tlx3也参与调控冷痛和炎症性疼痛的产生。本研究揭示了瘙痒感受神经元的发育的部分分子机制,同时也证实了TRPV1在慢性瘙痒产生中的重要性,为以后慢性瘙痒的治疗提供了理论依据。
[Abstract]:Itching and pain are two important senses of the body, which are conservative in the evolutionary process of the species. Under physiological conditions, the transient itching and pain is a protective mechanism that can prompt the harmful stimuli of the body to react to avoid further injury, such as pain mainly causing a retraction reaction, Itching causes strong scratching desire. Although the two are very different in personal experience and form of reaction, but they are not completely independent, there is a close relationship between the two. Some ion channels and receptors associated with pain also mediate the transfer of itching information. With the gradual deepening of itching research, itching related receptors And the pathway has gradually been found that the transfer of these receptors involved in itching information also requires the involvement of partial pain related ion channels, such as the inhibition of TRPA1 and TRPV1. pain and pruritus, and more and more evidence suggests that pain does inhibit itching, and a number of related inhibition pathways are also reported. The study of pain, itching has been a great progress since this century, although some of the pruritus related receptors, ion channels, neurotransmitters and neurons have been discovered, but the expression of these molecules and the regulatory mechanism of the related neuron development are not yet clear. Some reports have reported that some of them have been transferred. Recording factors are involved in the development and regulation of sensory neurons. Our laboratory work indicated that the transcription factor Tlx3 can regulate the differentiation of Trk A+ neurons in the dorsal root ganglia to the Ret+ neurons, and can also control the expression of a series of pain related molecules. As pruritus neurons are derived from Trk A+ neurons, we guessed that Tlx3 can also regulate the differentiation and development of pruritus neurons and the expression of pruritus related molecules. In order to explore this problem, we constructed a Tlx3 conditioned knockout mouse Tlx3F/F; Nav1.8-cre, which selectively knocks off Tlx3. in the Nav1.8+ neurons of the dorsal root ganglia to investigate whether the transcription factor Tlx3 regulates pruritus related points. We first compared the transcriptional groups of the dorsal root ganglion of Tlx3 conditioned knockout mice and control mice by RNAseq method, and found that most of the reported pruritus related molecules in the dorsal root ganglion of Tlx3 conditioned knockout mice were significantly decreased. In order to further verify the results of RNAseq, We used the m RNA in situ hybridization to detect the expression of some pruritus related molecules. We found that the expression of Mrgpr A3, IL31ra, Nppb, Sst in the dorsal root ganglion of Tlx3 conditioned knockout mice was completely missing. The results showed that Tlx3 was necessary for the expression of many pruritus related molecules. In order to explore the dorsal root of Tlx3 conditioned knockout mice Whether the down regulation of pruritus related molecules in the ganglia could affect the itching behavior of mice, we first compared the response of Tlx3 conditioned knockout mice and the control group to the acute itching induced by a variety of itchy agents. We selected four different types of itching agents: Compound48/80, chloroquine, alpha -Me-5-HT, SLIGRL-NH2.Comp Ound48/80 can cause histamine dependent pruritus, and the three types of.Tlx3 conditioned knockout mice that cause unhistamine dependent pruritus decreased the acute pruritus caused by these four itching agents compared with the control group, indicating that Tlx3 can regulate the production of acute itching, including histamine dependent pruritus and non histamine dependent Because many pruritus related receptors also mediate chronic itching, we also detected the response to chronic itching in Tlx3 conditioned knockout mice. We used two chronic pruritus models: Dry skin and allergic contact dermatitis (Allergic contact dermatitis, ACD) model.Tlx3 conditioned knockout mice The response to chronic itching caused by Dry skin was weaker than that in the control group, but the response to chronic itching caused by ACD was not significantly different from that of the control group. It indicated that Tlx3 was also involved in the regulation of some types of chronic pruritus. The expression of many itching related molecules was obviously defective, but the Tlx3 conditional knockout mice were still out. The obvious itching suggests the complexity of chronic itching and the involvement of other signaling pathways. Previous reports suggest that the expression of TRPV1 in some chronic pruritus models is up-regulated, suggesting that it may be involved in chronic itching. We found that the TRPV1 antagonist AMG9810 in Tlx3 conditioned knockout mice and control mice can significantly alleviate D Chronic itching caused by ry skin and ACD indicates that TRPV1 does participate in chronic pruritus. Pain can inhibit itching, and there is a report that TRPV1+/TRPA1+ neurons in the dorsal root ganglion are involved in this inhibition, and to explore whether it is also regulated by Tlx3, we use capsaicin (capsaicin) and AITC (allyl isothiocyanate) facial injection experiments. We found that facial injection of AITC in both Tlx3 conditioned knockout mice and control mice caused only obvious pain responses, and almost no pruritus, indicating that the inhibitory pathway was not significantly affected. However, the facial injection of capsaicin in Tlx3 conditioned knockout mice resulted in significantly enhanced pain responses, and more unexpectedly at the same time. In view of the inhibition pathway is normal, we think this obviously enhanced itching is due to capsaicin induced itching itself, and can not be completely suppressed by inhibition of the pathway. Capsaicin causes significantly enhanced pain and itching in the Tlx3 conditioned knockout rats. So we detected the expression of capsaicin receptor TRPV1 and found that the expression was significantly increased in the dorsal root ganglion, spinal cord and skin of Tlx3 conditioned knockout mice. In order to understand the expression pattern of TRPV1 in the dorsal root ganglion of Tlx3 conditioned knockout mice, we conducted a series of immunofluorescence experiments. Only about 27%Tlx3+ neurons express TRPV1 in the dorsal root ganglion of wild type mice and the overwhelming majority of TRPV1+ neurons do not express or low expression of Tlx3. It is further confirmed that Tlx3 can inhibit TRPV1 expression in the dorsal root ganglion of.Tlx3 conditioned knockout mice, which greatly expand to the IB4+ non peptide neurons, and it is also found in the dorsal root ganglion of.Tlx3 conditioned knockout mice. The proportion of CGRP+ peptide neurons also increased. Similarly, in the dorsal horn of the spinal cord of Tlx3 conditioned knockout mice, the projection area of the TRPV1+ nerve fiber extended to the IB4+ nerve fiber projection area, the two layer (lamina IIi). Considering that Tlx3 can regulate many pain related bodies, TRPV1 is also the main receptor for the sensation of heat stimulation. One of these, so we conducted a series of painful behavioural experiments to explore whether Tlx3 regulates the production of pain. We have found that the response of Tlx3 conditioned knockout mice to cold and inflammatory pain is significantly lower, while the response to mechanical, thermal and neuropathic pain is normal. Because TRPV1 is now known. The main receptor of the sensation of heat, although its expression level was significantly increased in the Tlx3 conditioned knockout mice, but the response of Tlx3 conditioned knockout mice to heat stimulation did not change significantly. In order to further verify this phenomenon, we used the spinal neuron activation experiment to determine whether the heat stimulation could be reduced in Tlx3 conditionality. More neurons were activated in the dorsal horn of the rat's spinal cord. The results showed that the number of neurons activated in the spinal cord of the Tlx3 conditioned knockout mice and the control group was not significantly different, but the number of neurons activated in the spinal cord of Tlx3 conditioned knockout mice was significantly more than that of the control group, indicating that the Tlx3 conditionality was knocked out. The increased sensitivity of TRPV1 to capsaicin in mice increased its sensitivity to capsaicin, but did not affect its response to thermal stimulation. In this study, this study confirmed that the transcription factor Tlx3 can regulate the expression of most pruritus related molecules, whose function loss causes the expression of pruritus related molecules to descend or disappear, thus causing acute itching and part of the mice to the Ministry. The response to chronic pruritus significantly weakened the expression of.Tlx3, which inhibited the expression of TRPV1, and the expression of TRPV1 in Tlx3 conditioned knockout mice increased significantly, which resulted in the increased pain and itching of the capsaicin in Tlx3 conditioned knockout mice, which also involved the regulation of cold pain and inflammatory pain. The molecular mechanism of the development of pruritus sensory neurons also confirms the importance of TRPV1 in the production of chronic itching and provides a theoretical basis for the treatment of chronic itching.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R338

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