本文選題：GPR30 + 肌萎縮側(cè)索硬化　； 參考：《河北醫(yī)科大學》2014年碩士論文

【摘要】：目的：肌萎縮側(cè)索硬化（amyotrophic lateral sclerosis，ALS）選擇性累及大腦皮層、腦干和脊髓前角的運動神經(jīng)元，病發(fā)后肌肉進行性萎縮、無力，多于發(fā)病后2到5年內(nèi)死于呼吸衰竭，是一種漸進性神經(jīng)系統(tǒng)變性疾病。約90%~95%的ALS為散發(fā)性，稱為散發(fā)性肌萎縮側(cè)索硬化（Spradicamyotrophic lateral sclerosis SALS），目前發(fā)病機制不清，多認為與氧化損傷、氨基酸興奮性性毒性、凋亡、線粒體損傷、軸漿轉(zhuǎn)運異常、細胞骨架異常等有關，但是，每一種假說都不能完全詮釋其發(fā)病過程；另5%~10%的ALS為家族性(Fimily amyotrophic lateral sclerosis FALS)，認為其中20%的發(fā)病與SOD1基因突變有關，而且SOD1G93A基因突變模型是目前應用最廣的ALS研究在體動物模型。 多年來的流行病學研究發(fā)現(xiàn)ALS發(fā)病存在明顯的性別差異：男性發(fā)病率大于女性，并且該差異隨年齡增長而減小[1]，女性比男性發(fā)病年齡相對較晚[2]。這提示ALS發(fā)病的性別差異或許源于雌激素(Estrogen, E)對神經(jīng)元的保護作用。 雌激素(Estrogen, E)為甾體類固醇性激素，文獻報道E的靶器官包括中樞神經(jīng)系統(tǒng)[3-6]。并且有研究證明E是一種神經(jīng)保護因子[7-9]，在許多神經(jīng)變性疾病模型中具有促進神經(jīng)元生存，保護組織完整性的作用[10-12]。越來越多的證據(jù)支持E在大腦的調(diào)節(jié)作用及其在年齡增長過程中維持大腦功能的重要性。年齡相關的E水平下降對大腦功能有不良影響，激素的減少與神經(jīng)變性疾病的進展有關。 雌激素的生物學功能大部分由經(jīng)典的雌激素核受體ERα/β通過調(diào)節(jié)基因轉(zhuǎn)錄來介導[13-14]，通常要數(shù)小時到數(shù)天起效，是經(jīng)典的慢速基因通路。研究發(fā)現(xiàn)ERα/β主要分布在脊髓后角外層[15-18]，而在前角運動神經(jīng)元幾乎沒有ERα/β的表達。大量研究發(fā)現(xiàn)，E2可以在數(shù)分鐘甚至數(shù)秒內(nèi)發(fā)揮改善血供、抗化、抗興奮性毒性的作用，且為非ERα/β位點依賴型。這提示除了經(jīng)典的慢速基因通路，E2還可以經(jīng)非基因快速通路起效。繼而有文獻報道E可以通過膜受體G-protein coupled receptor30(GPR30)發(fā)揮其快速非基因作用[19]。兩個獨立的研究機構分別發(fā)現(xiàn)GPR30（又稱GPER1）是一種新型雌激素受體[20-22]，并在中樞神經(jīng)系統(tǒng)廣泛分布[23-27]。近來的兩項實驗研究報道GPR30在脊髓自主神經(jīng)系統(tǒng)和神經(jīng)節(jié)有表達[28-29]。更有研究發(fā)現(xiàn)GPR30在脊髓前角運動神經(jīng)元有表達，這項研究認為GPR30有可能介導了E2對于運動神經(jīng)元的保護作用，并且發(fā)現(xiàn)雌激素不僅能通過激活GPR30發(fā)揮對運動神經(jīng)元的保護作用，還可能通過上調(diào)GPR30的表達而增強其作用[30]。 SOD1G93A轉(zhuǎn)基因小鼠（ALS小鼠）是FALS動物模型，其特征性臨床表現(xiàn)為成年（12周左右）發(fā)病，后肢運動功能障礙并逐漸進展至終末期（17-20周）[31]。而有關GPR30在ALS小鼠脊髓表達情況的報道少見。 本實驗應用SOD1G93A轉(zhuǎn)基因小鼠，觀察GPR30在ALS小鼠癥狀前期，癥狀早期和終末期腰段脊髓的表達特征，旨在探討性激素（sexhormon）與ALS發(fā)病中的關系，為進一步研究奠定理論依據(jù)。實驗內(nèi)容分為三部分： 第一部分GPR30在不同時期ALS小鼠腰段脊髓前角運動神經(jīng)元的表達 方法： 1模型建立及分組 飼養(yǎng)繁殖SOD1G93A轉(zhuǎn)基因雄性小鼠，參照韋爾切利1-5分評分法,發(fā)病時為4分，死亡時為1分,60天為癥狀前期，4分為癥狀早期，1分為終末期[32]。各對照組為同窩、同月齡不攜帶突變SOD1基因的小鼠。每組各3只小鼠。 2取材 應用10%水合氯醛350mg/kg腹腔注射麻醉后，4%多聚甲醛心臟灌注固定20min，取小鼠脊髓腰膨大，分別以4%多聚甲醛固定48h。 3免疫組化染色 后固定的組織塊震蕩切片25μm厚，應用免疫組織化學的方法觀察GPR30在ALS小鼠各時期腰段脊髓的表達分布特征。計數(shù)脊髓兩側(cè)前角GPR30免疫陽性α運動神經(jīng)元數(shù)量,并觀察脊髓前角有無GPR30免疫陽性的星形膠質(zhì)細胞。脊髓前角α運動神經(jīng)元數(shù)量的計數(shù)方法：對連續(xù)切片的每第五張切片的雙側(cè)脊髓前角的α運動神經(jīng)元計數(shù)，每段腰髓觀察10個切片。被計數(shù)的α運動神經(jīng)元標準為：位于前角、直徑＞25μm，細胞核清楚[33]。星形膠質(zhì)細胞的認定標準為：位于前角，形狀不規(guī)則，細胞核清楚，細胞突起粗短，分支多[34]。 4統(tǒng)計學處理 應用SPSS13.0統(tǒng)計學軟件對所得數(shù)據(jù)進行統(tǒng)計處理。采用均數(shù)標準差表示腰段脊髓切片兩側(cè)脊髓前角α運動神經(jīng)元計數(shù)之和，三組數(shù)據(jù)的比較采用單因素方差分析，兩組數(shù)據(jù)的比較采用兩個獨立樣本比較的t檢驗，以α=0.05為顯著性檢驗標準。 結(jié)果: 1SOD1G93A轉(zhuǎn)基因小鼠的鑒定 子代鼠的基因組DNA經(jīng)過PCR擴增之后、在GBOX-HR全自動凝膠成像系統(tǒng)下其瓊脂糖凝膠電泳結(jié)果示（Fig.1）：SOD1G93A轉(zhuǎn)基因陽性小鼠PCR產(chǎn)物條帶位于200-300bp之間（236bp）。沒有此條帶為非mSOD1的PCR產(chǎn)物，為非SOD1G93A轉(zhuǎn)基因鼠，即陰性對照小鼠。 2GPR30免疫組化 2.1ALS小鼠在癥狀前期、發(fā)病期、終末期腰段脊髓前角GPR30免疫陽性α運動神經(jīng)元計數(shù)分別為17.874.10、11.932.94、2.870.83，隨著ALS病情進展，免疫陽性α運動神經(jīng)元數(shù)目逐漸減少，到終末期僅見極少數(shù)殘存α運動神經(jīng)元，且各時期間均有顯著性差異（P＜0.05）；對照組各時期α運動神經(jīng)元計數(shù)分別為22.934.62、23.133.99、19.83.41，各時期間無顯著性差異（P＞0.05）；各時期ALS小鼠GPR30免疫陽性α運動神經(jīng)元數(shù)目與對照組相比均減少，且有顯著性差異（Table1，，Graph1）（Fig.2200×）。 2.2從分布部位看：對照組GPR30的表達在脊髓前角神經(jīng)元胞漿內(nèi)較均勻表達，核周圍著色較深；而轉(zhuǎn)基因鼠脊髓前角神經(jīng)元GPR30著色不均，呈顆粒狀深染，形成包涵體樣的結(jié)構；而且，與對照組不同，可見著色的星形膠質(zhì)細胞（Fig.2200×，F(xiàn)ig.3400×）。 結(jié)論: GPR30在脊髓前角神經(jīng)元有表達，主要在胞漿內(nèi)表達；隨病情進展，ALS轉(zhuǎn)基因鼠GPR30免疫陽性運動神經(jīng)元數(shù)量明顯減少，著色不均，呈顆粒狀深染，形成類包涵體樣結(jié)構，并且出現(xiàn)GPR30陽性的星形膠質(zhì)細胞。 第二部分GPR30在ALS小鼠脊髓前角細胞分布特征 方法： 1取60天齡SOD1G93A轉(zhuǎn)基因雄性小鼠及其對照組各3只。 2取材 10%水合氯醛350mg/kg腹腔注射麻醉后，心臟灌注4%多聚甲醛固定20min，取小鼠脊髓腰膨大，分別以4%多聚甲醛固定48h。 3免疫熒光染色 后固定的組織塊震蕩切片30μm厚，應用免疫熒光三標染色的方法在激光共聚焦顯微鏡下觀察GPR30在ALS小鼠腰段脊髓前角細胞的分布特征。 結(jié)果： 通過特異性免疫熒光三標的方法，我們發(fā)現(xiàn)GPR30在ALS小鼠脊髓前角α運動神經(jīng)元、星形膠質(zhì)細胞和小膠質(zhì)細胞均有特異性表達。在對照組，GPR30主要在α運動神經(jīng)元和小膠質(zhì)細胞表達，星形膠質(zhì)細胞也存在GPR30陽性表達，但數(shù)目極少（Fig.4,5）。 結(jié)論： GPR30在SOD1G93A轉(zhuǎn)基因鼠呈現(xiàn)星形膠質(zhì)細胞的異常表達。第三部分GPR30在不同時期SOD1G93A轉(zhuǎn)基因鼠星形膠質(zhì)細胞的表達。 方法： 1取第一部分所固定各組小鼠的脊髓腰膨大。 2免疫熒光染色 后固定的組織塊震蕩切片30μm厚，應用免疫熒光三標染色的方法在激光共聚焦顯微鏡下觀察GPR30在ALS小鼠腰段脊髓星形膠質(zhì)細胞的表達。 結(jié)果： 隨病情進展，SOD1G93A轉(zhuǎn)基因鼠GPR30免疫陽性星形膠質(zhì)細胞數(shù)量明顯增多,熒光強度明顯增強。而對照組各時期GPR30免疫陽性星形膠質(zhì)細胞都很少，熒光強度無明顯增強（Fig.6-11）。 結(jié)論: 隨病情進展，SOD1G93A轉(zhuǎn)基因鼠GPR30免疫陽性星形膠質(zhì)細胞數(shù)量明顯增多，熒光強度明顯曾強。
[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
Method錛

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