【作者】 于挺敏；

【导师】 张丽萍；

【作者基本信息】 东北师范大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 [目的]偏头痛是神经科常见的疾病,其发病机制至今尚不完全明确。随着对神经肽的深入研究,发现多种神经肽与偏头痛发生有关,如P物质、降钙素基因相关肽、神经激肽A等。其中,P物质作为神经递质在感觉信息传递和痛觉调制中的作用越来越多地引起人们的注意。目前的研究认为P物质对痛觉信息的传递可能起双重作用:一方面作为伤害信息传入纤维的神经递质,在脊髓背角、三叉神经脊束核等部位对痛觉传递的第一级突触起促进或易化作用;另一方面在脑内则发挥镇痛作用,对痛觉感受进行调制,降低对痛觉的敏感度。在与偏头痛相关的脑区,如中脑导水管周围灰质、中缝核等处存在丰富的P物质及其受体,然而,对在脑内的定量表达很少报道。实时定量PCR技术是目前国际公认的最准确、重复性最好的核酸分子定量定性检测方法,本研究建立了大鼠P物质及其受体的实时定量PCR检测方法,对偏头痛大鼠中脑P物质及其受体的基因表达进行绝对定量检测,同时应用免疫组织化学方法,检测该部位P物质及其受体蛋白水平的表达变化,探讨P物质及其受体在偏头痛病理生理过程中的变化情况。镁是人体必需的微量元素,它可以影响众多细胞功能,在细胞内起重要作用。临床应用镁剂预防、治疗偏头痛发作,取得了较为满意的效果。目前研究推测镁离子可能通过促进皮层扩散抑制、调节神经递质释放、改变脑兴奋性、影响血小板功能等多种途径参与偏头痛病理生理过程。本研究观察硫酸镁干预下偏头痛大鼠行为学、中脑P物质及其受体表达的变化。进一步证实硫酸镁对偏头痛的防治作用,同时探讨其机制。本研究拟解决如下问题:①观察偏头痛大鼠中脑P物质及其受体表达有何变化,进而探讨P物质与偏头痛发作的关系。②动物模型进一步证实硫酸镁干预后偏头痛大鼠行为学变化,并观察其变化是否存在剂量依赖性。③明确硫酸镁对偏头痛的影响机制是否与中脑P物质及其受体有关。[方法] 1.动物分组、造模与干预:将成年健康Wistar大鼠72只按随机数字表法分6组:对照组、偏头痛组、低剂量硫酸镁治疗组、高剂量硫酸镁治疗组、低剂量硫酸镁对照组、高剂量硫酸镁对照组、每组12只。对照组、低剂量硫酸镁对照组、高剂量硫酸镁对照组大鼠皮下注射生理盐水2mL/kg,偏头痛组、低剂量硫酸镁治疗组、高剂量硫酸镁治疗组大鼠皮下注射硝酸甘油10mg/kg,建立实验性偏头痛模型,以出现双耳发红、前肢频繁搔头、爬笼次数增多、咬尾、往返运动等提示模型动物头部不适的症状为造模成功的指标。5min后再分别给低剂量硫酸镁治疗组、低剂量硫酸镁对照组大鼠硫酸镁100mg/kg腹腔注射,高剂量硫酸镁治疗组、高剂量硫酸镁对照组大鼠硫酸镁300mg/kg腹腔注射。2.行为学观察:观察偏头痛组、低剂量硫酸镁治疗组、高剂量硫酸镁治疗组大鼠给予硝酸甘油后及对照组、低剂量硫酸镁对照组、高剂量硫酸镁对照组大鼠给予生理盐水后60min-90min内挠头、爬笼、咬尾、往返运动的次数并计数(每个症状出现1次计1分)。3.实时定量PCR检测各组大鼠中脑P物质及其受体的表达:偏头痛组、低剂量硫酸镁治疗组、高剂量硫酸镁治疗组大鼠给予硝酸甘油后及对照组、低剂量硫酸镁对照组、高剂量硫酸镁对照组大鼠给予生理盐水后2.0h,10 %水合氯醛(0.3 mL/ 100 g)麻醉大鼠,断头取脑。分离出中脑后立即置于液氮中,保存于-70℃冰箱中。取中脑组织50-100mg,根据总RNA提取试剂盒步骤操作,提取总RNA。经凝胶电泳后,使用紫外分光光度仪检测抽提总RNA的质量和浓度。要求A260/A280比值1.9-2.0,并计算RNA浓度(RNA样本浓度(μg/μL)= A260×稀释倍数×40÷1000)。每一标本取总RNA 300ng,进行反转录。合成好的cDNA置-70℃保存备用。应用SP、NK1受体和NK3受体特异引物,进行PCR扩增。PCR扩增产物经琼脂糖凝胶电泳,试剂盒回收。回收纯化的目的基因与pMD-18T载体连接,转化到E.coli DH5α感受态细胞中,氨苄青霉素筛选后提取质粒,重组质粒经过酶切及测序鉴定后,证实SP、NK1受体和NK3受体cDNA全长完全正确。将所提取的质粒在260nm下测OD260值,计算拷贝数,用无菌水10倍系列稀释、分装、作为标准品,-20℃保存。将各组大鼠中脑SP、NK1受体和NK3受体cDNA与相应重组质粒同时行SYBR GreenⅠ实时定量PCR扩增,计算各组大鼠中脑SP、NK1受体和NK3受体mRNA的绝对表达量。4.免疫组织化学检测各组大鼠中脑导水管周围灰质P物质及其受体的分布及表达:偏头痛组、低剂量硫酸镁治疗组、高剂量硫酸镁治疗组大鼠给予硝酸甘油后及对照组、低剂量硫酸镁对照组、高剂量硫酸镁对照组大鼠给予生理盐水后2.0h,各组大鼠用10%水合氯醛(300mg/kg)麻醉成功后,打开胸腔,暴露心脏,用小剪刀剪开右心耳,从左心室插管用生理盐水进行灌流,之后缓慢注入10%甲醛l00ml固定,断头取脑。将完整的鼠脑放入4%多聚甲醛中固定,常规脱水透明,低温石蜡包埋,切片,应用SP、NK1受体、NK3受体单克隆抗体行免疫组化染色。光镜下可见SP、NK1受体、NK3受体免疫反应阳性神经元的胞体和突起被均匀染成棕色。于400倍光镜下记录滑车神经核水平中脑导水管周围灰质腹侧区、背侧区各3个视野,计算平均阳性细胞数。5.用SAS统计学软件处理数据,各组大鼠行为学计分结果(挠头、爬笼、咬尾、往返运动的计分总和)用均数±标准差( x±S)表示,各组做方差分析,P<0.05表示差异有统计学意义。检测出的各组SP mRNA拷贝数变异较大,结果采用均数±标准差( x±S)表示,各组做方差分析,P<0.05表示差异有统计学意义。免疫组化检测出的各组大鼠中脑导水管周围灰质腹侧区、背侧区SP、NK1受体、NK3受体阳性细胞数,结果采用均数±标准差( x±S)表示,各组做方差分析,P<0.05表示差异有统计学意义。[结果] 1.行为学结果:低剂量硫酸镁治疗组和高剂量硫酸镁治疗组行为学改变较偏头痛组明显减轻(P<0.05),而高剂量硫酸镁治疗组大鼠行为学减轻程度较低剂量硫酸镁治疗组更为明显(P<0.05)。2.各组大鼠中脑SP、NK1受体和NK3受体基因水平表达情况:偏头痛组大鼠中脑P物质mRNA拷贝数明显低于对照组(P<0.05);低剂量硫酸镁治疗组、高剂量硫酸镁治疗组、低剂量硫酸镁对照组、高剂量硫酸镁对照组大鼠中脑P物质mRNA拷贝数明显高于偏头痛组和对照组(P<0.05)。高剂量硫酸镁治疗组、高剂量硫酸镁对照组大鼠中脑NK1受体mRNA拷贝数明显高于偏头痛组和对照组(P<0.05)。对照组、偏头痛组、低剂量硫酸镁治疗组、高剂量硫酸镁治疗组、低剂量硫酸镁对照组、高剂量硫酸镁对照组大鼠中脑NK3受体mRNA拷贝数无明显差异(P>0.05)。3.各组大鼠中脑导水管周围灰质SP、NK1受体和NK3受体蛋白水平表达情况:高剂量硫酸镁对照组大鼠中脑PAG区滑车神经核水平SP阳性细胞数明显高于对照组(P<0.05)。低剂量硫酸镁治疗组、高剂量硫酸镁治疗组大鼠中脑PAG区滑车神经核水平SP阳性细胞数明显高于偏头痛组和对照组(P<0.05)。低剂量硫酸镁治疗组、高剂量硫酸镁治疗组、低剂量硫酸镁对照组、高剂量硫酸镁对照组大鼠中脑PAG区滑车神经核水平NK1受体阳性细胞数明显高于偏头痛组和对照组(P<0.05)。对照组、偏头痛组、低剂量硫酸镁治疗组、高剂量硫酸镁治疗组、低剂量硫酸镁对照组、高剂量硫酸镁对照组大鼠中脑PAG区滑车神经核水平NK3受体阳性细胞数无明显差异(P>0.05)。[结论] 1.本研究所建立的检测大鼠P物质及其NK1受体、NK3受体mRNA的SYBR GreenⅠ实时定量PCR方法具有敏感性高、特异性强和线性范围广等特点,适用于对大鼠各种组织P物质及其NK1受体、NK3受体mRNA的大量样本检测。2.硝酸甘油10mg/kg皮下注射可以成功建立硝酸甘油型偏头痛大鼠模型。该模型行为症状表现(耳红、挠头、爬笼)及病理生化变化与人类偏头痛发作时的表现及变化有一定的相似性,符合模型设计时应遵循的标准化、相似性、重复性、适用性、经济性原则,且造模方法简单、经济,机制单一,可在短期内大量复制,是研究偏头痛较为理想的动物模型。在今后的研究中要进一步完善该模型行为学评价体系,制定统一定量的模型成功标准。3.偏头痛大鼠中脑SP基因水平表达减少,而SP蛋白水平表达未见明显变化,需进一步观察偏头痛不同时期SP的表达,明确SP在偏头痛发作过程中的变化规律。4.硫酸镁可以减轻偏头痛大鼠行为学症状,并具有剂量依赖性。5.硫酸镁治疗偏头痛的作用与促进中脑SP及NK1受体表达有关。6.硫酸镁预防偏头痛的作用,也可能与其增加中脑SP及NK1受体的表达有关。7. NK3受体不是SP的特异受体,它与偏头痛发作以及硫酸镁防治偏头痛的作用无明确关系。更多还原

【Abstract】 [Objective] With the increasing number of studies on neuropeptide, several kinds of neuropeptide such as substance P (SP), calcitonin-gene-related peptide and neurokinin A, have been proved to correlate with migraine. SP, as a neurotransmitter, plays an important role in sensory information transduction and pain modulation. Currently, SP is considered to elicit diphasic action on sensory information transduction, on one hand, SP is a neurotransmitter of wounding signal introducing into fibers, promoting or facilitating the first synapse of pain sense transmission in cornu dorsale medullae spinalis and spinal nucleus of trigeminal nerve; on the other hand, SP exhibits an analgesic effect in brains, modulates pain sense and reduce susceptibility to pain. Midbrain is of crucial significance in pain modulation, several researches addressing migraine show that numerous SP-positive and SP receptor-positive pericaryons and fibers are observed in periaqueductal gray and rapheal nuclei. However, no quantitative study reports SP mRNA in midbrain. Therefore, the present study adopts real-time quantitative PCR technique, which is considered as the most accurate, most repeatable and generally accepted detection method for nucleic acid molecular from a view of quantitation and qualitation, to conduct a absolutely quantitative study addressing SP mRNA in midbrain of migraine rats. At the same time , Detect the substance P and its receptor protein expression levels in the area with Immunohistochemistry, for discussion Of substance P and its receptor in the pathophysiology of migraine headache in the process of change.Magnesium allows for a better prevention and treatment of migraine by using magnesium in clinical practice. It is assumed that magnesium ion participates in pathophysiological process of migraine through such pathways as promoting cerebral cortex diffusion inhibition, modulating neurotransmitter release, altering cerebral excitability, and influencing platelet function. In the present study, experimental rat models of migraine was established with nitroglycerin by subcutaneous injection, so as to observe behavioral changes and SP and SP receptor expression in midbrain of migraine rats following magnesium intervention.The aim of this study was to investigate:①changes of SP expression in midbrain of migraine rats?②whether behavioral symptom of migraine rats was relieved or exhibited a dose-dependence following magnesium intervention?③whether magnesium inhibiting migraine correlates with SP?[Methods] 1. Grouping, model establishment and interventions : Totally 72 healthy adult Wistar rats were divided into six groups at random, namely control group, migraine group, low- and high-dose of magnesium sulfate treated groups, low- and high-dose of magnesium sulfate control groups, with 12 rats in each group. 2 mL/kg physiological saline was injected into rat cervical and back in the control group, low- and high-dose of magnesium sulfate control groups. Experimental migraine models were established through subcutaneous injection of 10 mg/kg nitroglycerin into rats in the migraine group, low- and high-dose of magnesium sulfate treated groups. The models were considered success upon the appearance of head complaint such as two ears reddish, frequently scratching head with forepaws, increasing number of climbing cage, biting tails, to and fro movement, etc.Five minutes following administration, rats in low dose groups were intraperitoneally injected with 100 mg/kg magnesium sulfate, while those in high dose groups with 300 mg/kg magnesium sulfate. No interventions were given in control group and migraine group.2. Behavioral observation : At 60-90 minutes following nitroglycerin injection, total number of appearing scratching head, climbing cage, biting tails, doing to and fro movement was measured (each symptom accounted for 1 point).3. Detect the substance P and its receptor mRNA expression with real-time PCR:Two hours following nitroglycerin injection, rats were anesthetized with 10% chloral hydrate (0.3 mL/100 g) and then decapitated. The midbrains was isolated and immediately placed in liquid nitrogen, preserved in a refrigerator at -70℃. 50-100 mg of midbrain tissues were added with 1 mL RNAiso Reagent, staying 5 minutes at room temperature, 4℃12 000 g centrifuged for 5 minutes, followed by the supernatant collection. Adding 0.2 mL chloroform, shaking and staying 5 minutes at room temperature, 4℃12 000 g centrifuged for 15 minutes, followed by the supernatant collection. Adding same volume of isopropyl alcohol, staying 10 minutes at room temperature, 4℃12 000 g centrifuged for 10 minutes, followed by the supernatant removal. Adding 1 mL 75% ethanol, 4℃12 000 g centrifuged for 5 minutes, followed by the supernatant removal. The precipitates were dissolved in 100μL RNA-free water. Subsequent to gel electrophoresis, total RNA quality and concentration were determined with ultraviolet spectrophotometer. The ratio of A260/A280 should maintain at 1.9-2.0, RNA concentration was calculated according to the formula: RNA concentration (μg/μL) = A260×attenuation multiple×40/1000. 20μL reaction system comprised RNA 300 ng with Reverse transcriptase. The synthetized cDNA was preserved at -70℃for later use. According to SP,NK1 receptor,NK3 receptor specific primer, the SP,NK1 receptor,NK3 receptor gene was synthetized with PCR apparatus. PCR amplification product was processed into 2% agarose gel electrophoresis, cutting target band, and recovered. The purified target gene was conjugated with pMD-18T carrier and transformed into E.coli DH5αcompetent cell. Subsequent to Ampicillin screening, plasmid extraction commenced. followed by restriction endonuclease HindⅢand BamHⅠdouble digestion, sequencing and identification. The absorbance value of the extracted plasmid at 260 nm was measured, copy number was also calculated. Following sterile water 10-fold serial dilution and subpackage, the standard specimen was preserved at -20℃. Different concentrations of plasmid standard specimens and synthetized cDNA was processed into quantitative PCR. The SP,NK1 receptor,NK3 receptor mRNA content in rat midbrain was calculated based on the melting curve.4. Detect the substance P and its receptor protein expression levels with Immunohistochemistry: Two hours following nitroglycerin injection, rats were anesthetized with 10% chloral hydrate (0.3 mL/100 g) and then Heart perfusion,Slow injection of 10% formaldehyde l00ml fixed,will be a complete ret brain into 4% paraformaldehyde fixed. Conventional dehydration transparent, Low-temperature paraffin-embeded, Sliced. Stained with SP, NK1 receptor, NK3 receptor monoclonal antibody。SP, NK1 receptor, NK3 receptor-Immunoreactive neurons in cell bodies and dendrites were evenly dyed brown。The level of trochlear nucleus in the ventral periaqueductal gray area of Ventral tegmental area and dorsal area in the three horizons, Calculation of the average number of positive cells.5. Statistical analysis: Data were statistically analyzed with SAS 6.12 software (SAS Software Institute, USA). Behavioral scores were expressed as mean±SD, and analysis of variance was applied for comparison among groups. SP mRNA cory number was expressed as mean±SD, and analysis of variance was applied for comparison among groups. The average number of positive cells of the level of trochlear nucleus in the ventral periaqueductal gray area of Ventral tegmental area and dorsal area in the three horizons, were expressed as mean±SD, and analysis of variance was applied for comparison among groups. A level of P < 0.05 was considered statistical significance.[Results] 1. Pain behavioral changes of experimental rats: Statistical results revealed that, nitroglycerin injection yielded to an obviously increment of behavioral scores in rats (P < 0.05). Compared with migraine group, the pain behavior was remarkably decreased in the low- and high-dose of magnesium sulfate treated groups (P < 0.05), especially in the high-dose (P < 0.05).2. The substance P and its receptor mRNA expression levels: SP ,NK1 receptor,NK3 receptor mRNA levels in rat midbrain was calculated according to the standard plot. Statistical analysis results showed that, the migraine group exhibited an obviously lower level of SP mRNA expression than control group (P < 0.05); Compared with migraine group and control group, the SP mRNA expression was enhanced following magnesium sulfate injection (P < 0.05). No significant differences were found among magnesium sulfate administrated groups (P > 0.05). Compared with migraine group and control group, the NK1 receptor mRNA expression was enhanced following high-dose magnesium sulfate administrated groups (P < 0.05). No significant differences were found with NK3 receptor mRNA expression among control group, migraine group, and magnesium sulfate administrated groups (P > 0.05). 3. The substance P and its receptor protein expression levels: Statistical analysis results showed that, Compared with control group, the SP expression was enhanced following high-dose of magnesium sulfate control group (P < 0.05), Compared with migraine group and control group, the SP expression was enhanced following magnesium sulfate injection (P < 0.05). Compared with migraine group and control group, the NK1 receptor expression was enhanced following magnesium sulfate administrated groups (P < 0.05). No significant differences were found with NK3 receptor expression among control group, migraine group, and magnesium sulfate administrated groups (P > 0.05). [Conclusion] 1. In the present study, SYBR GreenⅠreal-time quantitative PCR was successfully established to determine SP,NK1 receptor,NK3 receptor mRNA expression, the standard plot exhibits high linear correlation, sensitivity and repeatability, thus ensuring results more accurate and more reliable.2. Nitroglycerin has been generally acknowledged to establish an experimental animal model of migraine .the successful rat models are similar with the onset manifestations of migraine patients regarding behaviors (ear reddens, scratching head, climbing cage and so on) and pathological biomechanical changes. Model design was in accordance with the principles of standardization, similarity, reproducibility, applicability and economy. The models can be generously replicated in a short term. Therefore it is an optimal candidate model for migraine. However, this model has some limitations of behavioral observation, there is no a quantitative index to evaluate the success of model establishment, or scoring system for behavioral changes, therefore the assessment system on the behaviors should be further improved. 3. SP gene expression was reduced in midbrain when headache occurred.The levels of SP gene expression was decreased in brain of Migraine rat, while the SP was no significant change in protein level expression. we need to be further observed the expression of SP in different periods of migarine, clearly SP in migraine attacks in the process of change.4. varying doses of magnesium sulfate could relieve pain behavior in migraine rats and the therapeutic effect exhibited dose dependence, that is to say, high dose of magnesium sulfate resulted in obvious relieve of pain behaviors. 5. magnesium can cure migraine through the altered expression of SP and NK1 receptor in midbrain.6. magnesium sulfate may prevent migraine through the altered expression of SP and NK1 receptor in midbrain.7. NK3 receptor is not a specificity SP receptor. It is no clear relationship with migraineattacks,as well as the role of prevention and treatment of migraine attacks by magnesium sulfate.更多还原