三叉神经痛大鼠模型中CB1受体的表达变化

发布时间：2018-05-01 03:21

本文选题：三叉神经痛 + CB1受体　；参考：《南方医科大学》2012年硕士论文

【摘要】：1、背景大麻类植物是人类最早认识的成瘾性植物之一,使用大麻来止痛的记载可以追溯到数百年之前。最近十几年,随着内源性大麻素(endogenous cannabinoids, EC)的发现,以及两种内源性大麻素受体(cannabinoid type1receptor, CB1和cannabinoid type2receptor, CB2)的成功克隆,内源性大麻素系统(endocannabinoid system,ECS)的存在已经获得初步证实。CBl、CB2受体都属于G蛋白偶联受体,CBl受体由473个氨基酸,7个跨膜结构域构成。研究发现CBl受体主要分布于神经系统,以脑组织中表达为主,集中分布在大脑的海马、小脑和纹状体,多定位于神经末梢突触前膜,发挥调节神经递质释放的作用。在肾上腺,,心脏,肺,前列腺,脾脏和扁桃体等外周组织中也有微量表达。CB2受体由360个氨基酸,7个跨膜结构域构成,与CBl受体相反,CB2受体在中枢神经元中浓度很低,其主要在免疫系统中存在,包括脾脏、T细胞、扁桃体、B细胞及单核细胞中表达,主要发挥免疫调节作用,并能够抑制多种神经递质和细胞因子的释放。有研究表明CBl受体的拮抗剂可以增强对有害刺激的敏感性,且能够增强疼痛性神经元的活性。另外,CBl受体的激动剂可以抑制疼痛神经元的兴奋性,并且能够产生良好的镇痛效应。相关研究还表明CBl受体能够减少伤害性疼痛,从而抑制自发性疼痛的相关行为。原发性三义神经痛(Trigeminal neuralgia, TN)是一种神经慢性疾病,为三叉神经的分支分布区域里反复发作的刀割样阵发性的剧痛,其特点为三叉神经在口腔颌而部的某一分支或者几个支分支分布区域内突发的短暂而剧烈疼痛,并可长期的固定在某一分支,尤以第二、三支多见,亦可几支同时受累,持续疼痛时间为数秒钟到数分钟不等,而间歇期可以没有任何症状,其发作为自发性或由面部、口腔内轻微的触觉刺激所诱发,大多数是单侧发病,其中又以中老年人好发。目前,TN的发病机制尚不明确,确切的动物模型还难以建立。国内外学者大多认为TN的主要发病因素是周围因素,其中的血管压迫学说得到大多数学者的亲睐,所以,TN的动物模型基本上是通过眶下神经(infraorbital branch of the trigeminal nerve, ION)环扎术来模拟血管压迫现象,并且在ION支配区域的表面皮肤进行外在的机械性刺激,以此模拟TN扳机点来获得。三义神经脊束核(Spinal trigeminal caudal subnucleus, Vc)从外到内分为五层,在三叉神经脊束的内侧和延髓的外侧。Vc也是口腔颌面部组织的伤害性刺激向中枢传导的第一道门户,其尾侧亚核及其临近区域是躯体感觉的初级感受中枢,传递口腔颌面部的伤害性刺激信息。Vc的尾侧亚核在细胞构筑上相当于脊髓后角Ⅰ～Ⅵ层。Vc也被认为是面部伤害性传导通路的二级神经元所在区域,其与痛觉冲动的传递和调制有着密切的关系。Vc的Ⅱ层中胶状质中间神经元发生变性,便丧失了对传入的疼痛刺激的调节作用,失去了其对传入冲动的闸门作用,传入冲动可以很快达到一定的总和而引起疼痛剧烈发作。因此我们采用Vc组织来检测CBl受体的表达变化。有研究表明,损伤大鼠和猴子的外周神经,其存在于脊髓及背根神经节神经元中的μ阿片受体表达发生下降。而在坐骨神经(CCI)后的大鼠模型的同侧背角的表浅脊髓中CBl受体的表达同损伤大鼠单侧神经后的对侧丘脑区域内CBl受体的表达均发生上调。Nomura等和我们的研究团队的研究也都发现,对横断单侧下牙槽神经的大鼠鼻口部进行有害和无害的机械性刺激,则Fos和cdk5/p35蛋白样免疫反应性在其两侧Vc组织中均可被诱导,并且横断的对侧少于横断侧。Vc作为感觉中继站核,分别接受经三义神经传入的头而部感觉信息。坐骨神经CCI之后CBl受体表达发生上调的机制仍不明了,但是在该模型中,脊柱CBl受体的表达可能受到蛋白激酶C、酪氨酸激酶受体和相关的细胞内丝裂原活化蛋白激酶的调控。该模型的病理学特征与眶下神经慢性压迫性损伤(chronic constriction injury on the infraorbital branch of the trigeminal nerve, ION-CCI)大鼠模型的病理学特征相类似。综合上述的研究背景,本实验制备了三叉神经痛大鼠模型并检测了各组三叉神经痛大鼠模型Vc组织中CBl受体的表达变化。 2、目的我们假设TN大鼠模型的Vc组织中的神经元内存在CBl受体,因为目前仍无TN大鼠模型中CBl受体如何发挥相关作用方而的论述,此外,延髓下段的Vc组织中的神经元接受三叉神经的感觉神经传导的相关信息,所以,我们将TN的实验研究模型定为ION-CCI的大鼠模型,检验ION-CCI后大鼠Vc组织中CBl受体的表达变化,目的是为研究CBl受体与TN的临床治疗及致病机理的关联性打下实验基础。 3、材料与方法 3.1实验动物分组同一批次健康成体雄性SD大鼠,体重为300-350g,36只随机分为6组,每组6只。分为正常组、假手术组、ION-CCI1d组、ION-CCI3d组、ION-CCI7d组、ION-CCI14d组。雄性SD大鼠由南方医科大学珠江医院动物实验室以常规固体饲料饲养,温度控制在25℃左右,湿度在45%-50%之间,12h明暗循环(07：00—19：00)。为了消除因为动物行为的随意性对实验过程和结果的干扰,减少由此引起的假阳性结果,在实验开始之前每口08：00开始,用一自制塑料棒敲击笼壁四周和笼顶、触摸、提拿大鼠,于大鼠习惯之后,待大鼠平静时,用一自制毛刷刺激大鼠须垫部,3次/每侧,每次刺激需连续2次,两次刺激间隔不应少于30秒。两侧交替进行,直致大鼠从开始的抽鼻、探究、恐惧,甚至快速后退逃避、抓咬攻击等转为平静。 3.2暴露并行眶下神经结扎术实验动物腹腔内注射10%水合氯醛(350mg/kg),将麻醉效果满意的大鼠仰卧并固定其头部和四肢,沿着其右侧第一磨牙龈颊侧边缘,向其口鼻方向纵向切开长约1cm的切口,分离暴露ION及周围约5mm神经组织,在肉眼或显微镜下使用两根之间距离大约为2mmm的5.0铬制肠线疏松环扎ION,结扎ION的紧张度要求是减小ION的直径,使ION传导延缓,不可以完全阻滞神经表层血管的血液传导,血液循环必须通畅。术后6.0缝合线缝合伤口。假手术组大鼠除不结扎ION外,其他方面与手术组大鼠完全相同。所有操作均需在无菌环境下进行,手术前后无需使用任何抗生素。 3.3测得各实验组大鼠对电子VON FERY测痛仪所产生机械刺激的反应阈值。 3.4制备ION组织标本和Vc组织标本。分别称取各实验组大鼠的体重,按体重腹腔注10%水合氯醛(350mg/kg)麻醉后,取结扎区ION进行组织病理学观察(weil氏和HE染色)。Vc组织的定位按照Paxinos等制定的大鼠脑立体定位图谱进行定位,切取定好位后的Vc组织,将标记好后的各实验组大鼠的Vc组织放入冰箱保存(-80℃)。 3.5提取Vc组织抗原蛋白取出上述标记好的各实验组大鼠的Vc组织块用PBS液冲洗,将Vc组织放置于冰上用干净的剪刀尽量剪碎,再将其放入匀浆器内,每5mg加入300μl配制好的细胞裂解缓冲液后,裂解30min后,用移液枪移至1.5ml空离心管里,随后离心5min(12000rpm、低于4℃),将上清液取出之后小心放入已经准备好的空离心管里,然后置入冰箱冷冻储存(-20℃)。 3.6测定Vc组织上清液蛋白含量 3.7CBl受体的Western blotting检测 4、统计方法与统计软件采用SPSS13.0统计软件对本实验的数据进行统计学分析,使用均数±标准差(x±s)来表达实验研究结果,单因素方差分析(One-Way ANOVA)和重复测量方差分析(Repeated Measures)用于多组间比较,首先进行方差齐性检验,如方差不齐,多重比较采用Dunnett's进行检验；如方差齐性,多重比较使用Bonferroni进行检验；P0.05表示差异有统计学意义。两组间比较用配对样本t检验分析(Paired-Samples T Test),P0.05表示差异有统计学意义。 5、结果 (1)行为学观察结果显示,正常组和假于术组每天疼痛阈值比较并无显著差异,P均大于0.05。手术组和对照组比较术后疼痛阈值具有显著差异,P均小于0.05。 (2)组织学观察结果显示：正常结构消失,术后缩窄环区域神经纤维肿胀逐渐明显；受压迫神经纤维粗细不一致、分布不均匀、甚至发生变性,被环扎的神经轴突裸露、稀疏、变细、厚薄不一、髓鞘正常结构消失,呈现出虫蚀状节段性脱失和空泡样改变,连续性中断,大量出现许旺细胞。 (3)CBl受体在手术组(ION-CCI1d组、ION-CCI3d组、ION-CCI7d组和ION-CCI14d组)大鼠Vc组织中的含量表达依次上调,但正常组和假手术组大鼠Vc组织中CBl受体含量表达无显著性差异；假手术组大鼠手术侧与对侧Vc组织中CBl受体含量表达无差异：ION-CCl14d组手术侧与对侧Vc组织中CBl受体含量表达有显著差异。6、结论 (1)外周神经损伤可呈时间依赖的形式导致Vc组织中CBl受体表达上调。 (2) ION-CCI后诱导了其同侧和对侧CBl受体表达增加,在CCI侧CBl受体表达上调的程度显著大于其对侧。 (3)CBl受体表达变化与周围神经疼痛阈值变化无直接关联。
[Abstract]:1, the background
Cannabis is one of the earliest known addictive plants in human beings. The record of using cannabis to stop pain can be traced back to hundreds of years. In the last decade, with the discovery of endogenous endogenous cannabinoids (EC), and two endogenous endogenous receptors (cannabinoid type1receptor, CB1 and cannabinoid type2receptor, C) The existence of the successful cloning of B2, the existence of endocannabinoid system (ECS), has preliminarily proved.CBl, CB2 receptors are G protein coupled receptors, CBl receptors are composed of 473 amino acids and 7 transmembrane domains. The study found that CBl receptors are mainly distributed in the nervous system, mainly in brain tissue, concentrated in the brain. The hippocampus, cerebellum and striatum, mostly located at the nerve terminal presynaptic membrane, play a role in regulating the release of neurotransmitters. In the adrenal, heart, lung, prostate, spleen and tonsillar, the.CB2 receptors are also expressed in 360 amino acids and 7 transmembrane domains, contrary to the CBl receptor, and the CB2 receptor is in central neurons. The concentration is very low, which mainly exists in the immune system, including the spleen, T cells, tonsillar, B cells and mononuclear cells, which mainly play the role of immunoregulation, and can inhibit the release of various neurotransmitters and cytokines. Studies have shown that CBl receptor antagonists can enhance the sensitivity to harmful stimuli and enhance the pain. In addition, CBl receptor agonists can inhibit the excitability of pain neurons and produce good analgesic effects. Related studies also suggest that CBl receptors can reduce nociceptive pain and inhibit spontaneous pain related behavior.
Primary trigeminal neuralgia (Trigeminal neuralgia, TN) is a chronic neuropathic disease, a recurrent knife - cut paroxysmal pain in the branching area of the trigeminal nerve, characterized by transient and severe pain of the trigeminal nerve in the oral and maxillofacial branches or in several branches of the branch, and for a long time Fixed in a branch, especially second, third more common, also several simultaneous involvement, the duration of pain for a few seconds to several minutes, and the intermittent period can be without any symptoms, its hair as spontaneous or from the face, a slight tactile stimulation in the mouth, the majority of the onset of the onset, among them, the elderly good hair. Currently, TN The pathogenesis is still not clear, the exact animal model is still difficult to establish. Most scholars at home and abroad believe that the main factors of TN are the surrounding factors, and the theory of vascular compression has been favored by most scholars, so the animal model of TN is basically through the orbital nerve (infraorbital branch of the trigeminal nerve, ION) ring ligation. To simulate the phenomenon of vascular compression, and to conduct mechanical stimulation on the surface of the ION domination area to simulate the TN trigger point.
The Spinal trigeminal caudal subnucleus (Vc) is divided into five layers from outer to inner. In the medial and lateral medulla of the trigeminal spinal tract, the lateral.Vc is the first gateway to the center of the oral and maxillofacial tissue. The caudal subnucleus and its adjacent area are the primary sensory center of the somatosensory, passing the oral cavity. The nociceptive stimulus information of the maxillofacial region, the caudal subnucleus of.Vc, is equivalent to the posterior horn of the spinal cord in the cellular architecture, which is also considered to be the region of the two stage neurons of the facial nociceptive transmission pathway, which is closely related to the transmission and modulation of the algesia impulse and the degeneration of the gelatinous intermediate neurons in the layer II of.Vc. The modulation of the afferent pain stimulus has lost its effect on the afferent impulse, and the afferent impulse can quickly reach a certain sum and cause severe pain attacks. Therefore, we use Vc tissue to detect the changes in the expression of the CBl receptor.
Some studies have shown that the peripheral nerve of the injured rat and the monkey has a decrease in the expression of micron opioid receptor in the spinal and dorsal root ganglion neurons. The expression of the CBl receptor in the superficial spinal cord of the same lateral dorsal horn of the rat model of the sciatic nerve (CCI) and the expression of the CBl receptor in the lateral thalamus region after the injury of the unilateral nerve of the rat .Nomura and our research team also found that Fos and cdk5/p35 protein like immunoreactivity can be induced in both sides of the Vc tissue in the nasal mouth of the rat with unilateral inferior alveolar nerve, and the opposite side of the transection is less than the transverse.Vc as a sensory relay. The mechanism of the up regulation of CBl receptor expression after CCI is still unknown, but in this model, the expression of the CBl receptor in the spine may be regulated by protein kinase C, tyrosine kinase receptor and related intracellular mitogen activated protein kinase. Pathological features are similar to the pathological features of the rat model of chronic suborbital nerve compression (chronic constriction injury on the infraorbital branch of the trigeminal nerve, ION-CCI).
In this study, a rat model of trigeminal neuralgia was prepared and the changes in the expression of CBl receptor in the Vc tissues of three trigeminal neuralgia rats were detected in this experiment.
2, the purpose
We assume that the neurons in the Vc tissue of the TN rat model are stored in the CBl receptor, because there is still no discussion on how CBl receptors play a role in the TN rat model. In addition, the neurons in the Vc tissue of the lower medulla accept the related information of the sensory nerve conduction of the trigeminal nerve. So, we set the experimental model of the TN. For the rat model of ION-CCI, the changes in the expression of CBl receptor in the Vc tissues of rats after ION-CCI were examined. The aim is to lay a foundation for the study of the clinical treatment of CBl receptor and the association of the pathogenesis of TN.
3, materials and methods
3.1 group of experimental animals
The same batch of healthy adult male SD rats, with a weight of 300-350g, were divided into 6 groups randomly, with 6 rats in each group, which were divided into normal group, sham operation group, group ION-CCI1d, group ION-CCI3d, ION-CCI7d group, group ION-CCI14d. The male SD rats were fed by the laboratory animal laboratory of Zhujiang Hospital of Southern Medical University, the temperature was controlled at about 25, and the humidity was 4 Between 5%-50%, 12h and dark cycle (07:00 - 19:00). In order to eliminate the interference of the randomness of animal behavior to the experimental process and results, reduce the resulting false positive results. Before the start of the experiment, each 08:00 begins with a homemade plastic rod knocking around the cage wall and caged top, touching, lifting the rat, after the rat habit, to be big. When the rat was calm, a self-made brush was used to stimulate the rat's mattress. 3 times / each side, each stimulus should be 2 times, and the two time interval should not be less than 30 seconds. The two sides were alternately carried out, which directly caused the rat from the beginning of the nose, the inquiry, the fear, even the quick retreat escape, and the bite attack and other tranquility.
3.2 exposure and parallel supraorbital nerve ligation
The experimental animals were intraperitoneally injected with 10% chloral chloral (350mg/kg). The rats with satisfactory anesthesia were supine and fixed on the head and limbs. Along the edge of the cheek of the first molar of the right side, the incision was cut longitudinally for 1cm in the direction of the mouth and nose. The ION and the surrounding 5mm were separated, and the two between the eyes and the microscope were used between the eyes and the microscope. The 5 chromic intestinal line from about 2mmm was loosely ligated ION, the tension of ligation of ION was required to reduce the diameter of the ION, delay the ION conduction, not completely block the blood conduction of the blood vessels of the nerve surface, and the blood circulation must be smooth. The 6 suture line sutured the wound after operation. The rats in the sham operation group were in addition to the ligation of ION, and the other aspects were finished with the operation group. All operations are performed in sterile environment. No antibiotics are needed before and after operation.
3.3 the response threshold of each experimental group to the mechanical stimulus produced by the electronic VON FERY pain analyzer was measured.
3.4 the specimens of ION tissue and Vc tissue were prepared.
The body weight of the rats in the experimental group was taken respectively, and after anaesthesia was injected into the abdominal cavity of 10% chloral chloral (350mg/kg), the ION of the ligation area was taken for histopathological observation (Weil's and HE staining), and the location of the.Vc tissue in the rat brain was located according to Paxinos and so on, and the Vc tissue after the fixed position was cut and the experimental groups were marked. The Vc tissues of the rats were stored in the refrigerator (-80 C).
3.5 extraction of Vc tissue antigen protein
The Vc tissues of the rats in the experimental groups were flushed with PBS liquid, and the Vc tissues were placed on the ice with a clean scissors, and then put into the homogenizer. After each 5mg was added to the 300 mu l buffer solution, the cracking 30min was transferred to the 1.5ml centrifuge tube with a graving gun and then centrifuged 5min (12000rpm, less than 4). After removing the supernatant, carefully put it into the empty centrifuge tube and store it in the fridge for storage (-20 C).
3.6 determination of protein content in the supernatant of Vc tissue
Detection of 3.7CBl receptor by Western blotting
4, statistical method and statistical software
The data of this experiment were statistically analyzed with SPSS13.0 statistical software, and the experimental results were expressed using mean mean deviation standard deviation (x + s). Single factor variance analysis (One-Way ANOVA) and repeated measurement variance analysis (Repeated Measures) were used for multigroup comparison. First, the variance homogeneity test was carried out, such as variance inhomogeneous, multiple comparison using Dunn. Ett's was tested, such as homogeneity of variance, multiple comparison using Bonferroni to test, P0.05 indicated that the difference was statistically significant. The two groups were compared with the paired sample t test analysis (Paired-Samples T Test), P0.05 indicated that the difference was statistically significant.
5, the result
(1) the results of behavioral observation showed that there was no significant difference in the threshold of pain between the normal group and the false group. The P was greater than that in the 0.05. operation group and the control group, and the pain threshold was significantly different after the operation, and the P was less than 0.05..
(2) the histological observation showed that the normal structure disappeared and the nerve fiber swelling in the constrictive ring region was gradually obvious. The compressed nerve fibers were not uniform, uneven, even denatured, and the axons were exposed, thinned, thin, and thin, and the normal structure of myelin disappeared. Vacuolar changes, continuity interruption, a large number of Schwann cells.
(3) the content expression of CBl receptor in the Vc tissue of the operation group (group ION-CCI1d, ION-CCI3d, ION-CCI7d and ION-CCI14d) was up, but there was no significant difference in the expression of CBl receptor in the normal group and the sham operation group, and there was no difference in the expression of the CBl receptor content in the operative side and the contralateral Vc tissue in the sham operation group: ION-CCl1 There was a significant difference in the expression of CBl receptor between the operative side and contralateral Vc tissue in group 4D,.6
(1) peripheral nerve injury can be expressed in a time-dependent manner, resulting in up regulation of CBl receptor expression in Vc tissues.
(2) after ION-CCI, the expression of CBl receptor on ipsilateral and contralateral side was increased, and the expression of CBl receptor on CCI side was significantly higher than that in contralateral side.
(3) there was no direct correlation between the expression of CBl receptor and peripheral nerve pain threshold.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R-332

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