GPR30在家族性肌萎缩侧索硬化动物模型腰段脊髓中的表达特征

发布时间：2018-05-05 08:15

  本文选题：GPR30 + 肌萎缩侧索硬化　； 参考：《河北医科大学》2014年硕士论文

【摘要】：目的：肌萎缩侧索硬化（amyotrophic lateral sclerosis，ALS）选择性累及大脑皮层、脑干和脊髓前角的运动神经元，病发后肌肉进行性萎缩、无力，多于发病后2到5年内死于呼吸衰竭，是一种渐进性神经系统变性疾病。约90%~95%的ALS为散发性，称为散发性肌萎缩侧索硬化（Spradicamyotrophic lateral sclerosis SALS），目前发病机制不清，多认为与氧化损伤、氨基酸兴奋性性毒性、凋亡、线粒体损伤、轴浆转运异常、细胞骨架异常等有关，但是，每一种假说都不能完全诠释其发病过程；另5%~10%的ALS为家族性(Fimily amyotrophic lateral sclerosis FALS)，认为其中20%的发病与SOD1基因突变有关，而且SOD1G93A基因突变模型是目前应用最广的ALS研究在体动物模型。 多年来的流行病学研究发现ALS发病存在明显的性别差异：男性发病率大于女性，并且该差异随年龄增长而减小[1]，女性比男性发病年龄相对较晚[2]。这提示ALS发病的性别差异或许源于雌激素(Estrogen, E)对神经元的保护作用。 雌激素(Estrogen, E)为甾体类固醇性激素，文献报道E的靶器官包括中枢神经系统[3-6]。并且有研究证明E是一种神经保护因子[7-9]，在许多神经变性疾病模型中具有促进神经元生存，保护组织完整性的作用[10-12]。越来越多的证据支持E在大脑的调节作用及其在年龄增长过程中维持大脑功能的重要性。年龄相关的E水平下降对大脑功能有不良影响，激素的减少与神经变性疾病的进展有关。 雌激素的生物学功能大部分由经典的雌激素核受体ERα/β通过调节基因转录来介导[13-14]，通常要数小时到数天起效，是经典的慢速基因通路。研究发现ERα/β主要分布在脊髓后角外层[15-18]，而在前角运动神经元几乎没有ERα/β的表达。大量研究发现，E2可以在数分钟甚至数秒内发挥改善血供、抗化、抗兴奋性毒性的作用，且为非ERα/β位点依赖型。这提示除了经典的慢速基因通路，E2还可以经非基因快速通路起效。继而有文献报道E可以通过膜受体G-protein coupled receptor30(GPR30)发挥其快速非基因作用[19]。两个独立的研究机构分别发现GPR30（又称GPER1）是一种新型雌激素受体[20-22]，并在中枢神经系统广泛分布[23-27]。近来的两项实验研究报道GPR30在脊髓自主神经系统和神经节有表达[28-29]。更有研究发现GPR30在脊髓前角运动神经元有表达，这项研究认为GPR30有可能介导了E2对于运动神经元的保护作用，并且发现雌激素不仅能通过激活GPR30发挥对运动神经元的保护作用，还可能通过上调GPR30的表达而增强其作用[30]。 SOD1G93A转基因小鼠（ALS小鼠）是FALS动物模型，其特征性临床表现为成年（12周左右）发病，后肢运动功能障碍并逐渐进展至终末期（17-20周）[31]。而有关GPR30在ALS小鼠脊髓表达情况的报道少见。 本实验应用SOD1G93A转基因小鼠，观察GPR30在ALS小鼠症状前期，症状早期和终末期腰段脊髓的表达特征，旨在探讨性激素（sexhormon）与ALS发病中的关系，为进一步研究奠定理论依据。实验内容分为三部分： 第一部分GPR30在不同时期ALS小鼠腰段脊髓前角运动神经元的表达 方法： 1模型建立及分组 饲养繁殖SOD1G93A转基因雄性小鼠，参照韦尔切利1-5分评分法,发病时为4分，死亡时为1分,60天为症状前期，4分为症状早期，1分为终末期[32]。各对照组为同窝、同月龄不携带突变SOD1基因的小鼠。每组各3只小鼠。 2取材 应用10%水合氯醛350mg/kg腹腔注射麻醉后，4%多聚甲醛心脏灌注固定20min，取小鼠脊髓腰膨大，分别以4%多聚甲醛固定48h。 3免疫组化染色 后固定的组织块震荡切片25μm厚，应用免疫组织化学的方法观察GPR30在ALS小鼠各时期腰段脊髓的表达分布特征。计数脊髓两侧前角GPR30免疫阳性α运动神经元数量,并观察脊髓前角有无GPR30免疫阳性的星形胶质细胞。脊髓前角α运动神经元数量的计数方法：对连续切片的每第五张切片的双侧脊髓前角的α运动神经元计数，每段腰髓观察10个切片。被计数的α运动神经元标准为：位于前角、直径＞25μm，细胞核清楚[33]。星形胶质细胞的认定标准为：位于前角，形状不规则，细胞核清楚，细胞突起粗短，分支多[34]。 4统计学处理 应用SPSS13.0统计学软件对所得数据进行统计处理。采用均数标准差表示腰段脊髓切片两侧脊髓前角α运动神经元计数之和，三组数据的比较采用单因素方差分析，两组数据的比较采用两个独立样本比较的t检验，以α=0.05为显著性检验标准。 结果: 1SOD1G93A转基因小鼠的鉴定 子代鼠的基因组DNA经过PCR扩增之后、在GBOX-HR全自动凝胶成像系统下其琼脂糖凝胶电泳结果示（Fig.1）：SOD1G93A转基因阳性小鼠PCR产物条带位于200-300bp之间（236bp）。没有此条带为非mSOD1的PCR产物，为非SOD1G93A转基因鼠，即阴性对照小鼠。 2GPR30免疫组化 2.1ALS小鼠在症状前期、发病期、终末期腰段脊髓前角GPR30免疫阳性α运动神经元计数分别为17.874.10、11.932.94、2.870.83，随着ALS病情进展，免疫阳性α运动神经元数目逐渐减少，到终末期仅见极少数残存α运动神经元，且各时期间均有显著性差异（P＜0.05）；对照组各时期α运动神经元计数分别为22.934.62、23.133.99、19.83.41，各时期间无显著性差异（P＞0.05）；各时期ALS小鼠GPR30免疫阳性α运动神经元数目与对照组相比均减少，且有显著性差异（Table1，，Graph1）（Fig.2200×）。 2.2从分布部位看：对照组GPR30的表达在脊髓前角神经元胞浆内较均匀表达，核周围着色较深；而转基因鼠脊髓前角神经元GPR30着色不均，呈颗粒状深染，形成包涵体样的结构；而且，与对照组不同，可见着色的星形胶质细胞（Fig.2200×，Fig.3400×）。 结论: GPR30在脊髓前角神经元有表达，主要在胞浆内表达；随病情进展，ALS转基因鼠GPR30免疫阳性运动神经元数量明显减少，着色不均，呈颗粒状深染，形成类包涵体样结构，并且出现GPR30阳性的星形胶质细胞。 第二部分GPR30在ALS小鼠脊髓前角细胞分布特征 方法： 1取60天龄SOD1G93A转基因雄性小鼠及其对照组各3只。 2取材 10%水合氯醛350mg/kg腹腔注射麻醉后，心脏灌注4%多聚甲醛固定20min，取小鼠脊髓腰膨大，分别以4%多聚甲醛固定48h。 3免疫荧光染色 后固定的组织块震荡切片30μm厚，应用免疫荧光三标染色的方法在激光共聚焦显微镜下观察GPR30在ALS小鼠腰段脊髓前角细胞的分布特征。 结果： 通过特异性免疫荧光三标的方法，我们发现GPR30在ALS小鼠脊髓前角α运动神经元、星形胶质细胞和小胶质细胞均有特异性表达。在对照组，GPR30主要在α运动神经元和小胶质细胞表达，星形胶质细胞也存在GPR30阳性表达，但数目极少（Fig.4,5）。 结论： GPR30在SOD1G93A转基因鼠呈现星形胶质细胞的异常表达。第三部分GPR30在不同时期SOD1G93A转基因鼠星形胶质细胞的表达。 方法： 1取第一部分所固定各组小鼠的脊髓腰膨大。 2免疫荧光染色 后固定的组织块震荡切片30μm厚，应用免疫荧光三标染色的方法在激光共聚焦显微镜下观察GPR30在ALS小鼠腰段脊髓星形胶质细胞的表达。 结果： 随病情进展，SOD1G93A转基因鼠GPR30免疫阳性星形胶质细胞数量明显增多,荧光强度明显增强。而对照组各时期GPR30免疫阳性星形胶质细胞都很少，荧光强度无明显增强（Fig.6-11）。 结论: 随病情进展，SOD1G93A转基因鼠GPR30免疫阳性星形胶质细胞数量明显增多，荧光强度明显曾强。
[Abstract]:Objective: amyotrophic lateral sclerosis (ALS) selectively involves the motor neurons in the cerebral cortex, the brain stem and the anterior horn of the spinal cord. The muscle atrophy and weakness after the onset of the disease is more than 2 to 5 years after the onset of the disease. It is a progressive degenerative disease. The ALS of about 90%~95% is sporadic and is called sporadic. The pathogenesis of Spradicamyotrophic lateral sclerosis SALS (amyotrophic lateral sclerosis) is not clear at present. It is considered to be related to oxidative damage, amino acid excitotoxicity, apoptosis, mitochondrial damage, abnormality of axoplasm transport and cytoskeleton, but each hypothesis can not fully interpret its pathogenesis; the ALS of 5%~10% is ALS The familial (Fimily amyotrophic lateral sclerosis FALS) thought that 20% of the disease was associated with the mutation of the SOD1 gene, and the SOD1G93A gene mutation model was the most widely used ALS in the body animal model.
Many years of epidemiological studies have found that there is a significant gender difference in the incidence of ALS: the incidence of male morbidity is greater than that of women, and the difference decreases with the age of [1], and the age of women is relatively later than the age of male [2]., which suggests that the gender differences in ALS may be derived from the protective effect of estrogen (Estrogen, E) on neurons.
Estrogen (E) is steroid steroid hormone. It is reported that the target organ of E includes the central nervous system [3-6]. and there is a study that E is a neuroprotective factor [7-9]. In many neurodegenerative disease models, there is more and more evidence to promote the survival of neurons and protect the integrity of tissues by [10-12].. More and more evidence supports E in the brain. The regulatory role and the importance of maintaining brain function during the age of growth. The decline in age related E levels has an adverse effect on brain function, and the decrease of hormone is associated with the progression of neurodegenerative diseases.
The biological function of estrogen is mostly mediated by the classical estrogen receptor ER alpha / beta by regulating gene transcription to mediate [13-14]. It usually takes hours to several days to take effect and is a classic slow gene pathway. The study found that ER alpha / beta is mainly distributed in the outer layer of the spinal posterior horn of [15-18], and there is almost no expression of ER alpha / beta in the anterior horn motoneurons. Quantitative studies have found that E2 can play a role in improving blood supply, anti chemical, and excitability toxicity in a few minutes or even seconds, and is a non ER alpha / beta dependent type. This suggests that E2 can also take effect on non gene fast pathways in addition to the classic slow gene pathway. Then there is a literature report that E can be used by the membrane receptor G-protein coupled receptor30 (GPR30). GPR30 (also known as GPER1) is a new type of estrogen receptor [20-22], with its rapid non gene action [19]. two independent research institutions, and the two recent experimental studies on the widespread distribution of [23-27]. in the central nervous system reported that GPR30 has been more studied in the spinal autonomic nervous system and the divine scripture expression [28-29]. and found GPR30 in the spine. The premedullary motoneurons are expressed. This study suggests that GPR30 may mediate the protective effect of E2 on motoneurons, and that estrogen can not only protect the motor neurons by activating the GPR30, but also enhance the role of the GPR30 by increasing the expression of [30]..
SOD1G93A transgenic mice (ALS mice) are FALS animal models. Their characteristic clinical manifestations are adult (about 12 weeks), hindlimb movement dysfunction and progressing to end-stage (17-20 weeks) [31].. The expression of GPR30 in the spinal cord of ALS mice is rare.
In this experiment, SOD1G93A transgenic mice were used to observe the expression of GPR30 in the early stage of symptoms, early symptoms and end stage spinal cord in ALS mice. The purpose of this study was to explore the relationship between sex hormone (sexhormon) and the pathogenesis of ALS, and lay a theoretical basis for further research. The experimental contents were divided into three parts:
Part one GPR30 expression of motor neurons in lumbar spinal cord anterior horn of ALS mice at different stages
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