MPTP損傷對(duì)食蟹猴嗅球神經(jīng)遞質(zhì)表達(dá)的影響
發(fā)布時(shí)間：2018-08-15 15:17
【摘要】：研究目的 不同于傳統(tǒng)的限于運(yùn)動(dòng)系統(tǒng)認(rèn)知，帕金森病更可以視為是以嗅覺障礙為主要癥狀而以運(yùn)動(dòng)障礙為次要癥狀的疾病。作為嗅覺通路中重要樞紐的嗅球在該疾病中的病理改變亦被廣泛研究。MPTP非人靈長(zhǎng)類帕金森病模型是迄今最理想的研究帕金森病發(fā)病機(jī)制的動(dòng)物模型。 本研究旨在通過(guò)免疫組化的方法觀察食蟹猴嗅球神經(jīng)解剖和神經(jīng)化學(xué)的特征,包括:1.食蟹猴嗅球組織結(jié)構(gòu)學(xué)特點(diǎn)；2.食蟹猴嗅球各主要神經(jīng)遞質(zhì)的表達(dá)情況,膠質(zhì)細(xì)胞的分布；3.MPTP損傷對(duì)食蟹猴嗅球神經(jīng)遞質(zhì)表達(dá)，膠質(zhì)細(xì)胞和細(xì)胞凋亡的影響，MPTP損傷對(duì)食蟹猴嗅球中各主要神經(jīng)遞質(zhì)表達(dá)、凋亡信號(hào)、膠質(zhì)細(xì)胞的影響，這些知識(shí)可以給研究帕金森病嗅覺障礙積累一定的背景知識(shí)。 第一部分MPTP食蟹猴帕金森病模型的建立 方法 成年健康食蟹猴6只，在動(dòng)物中心嚴(yán)格按照AAALAC (Association forAssessment and Accreditation of Laboratory Animal Care，國(guó)際實(shí)驗(yàn)動(dòng)物評(píng)估和認(rèn)可管理委員會(huì)）規(guī)定和要求對(duì)動(dòng)物進(jìn)行日常的飼養(yǎng)、關(guān)懷和管理。采用小劑量連續(xù)給藥方法，，即0.2mg/kg MPTP-HCl溶液,于下肢靜脈緩慢注入，每天早晨1次（9AM）。采用個(gè)體化原則，以出現(xiàn)典型的臨床癥狀為注射終點(diǎn)。 結(jié)果 注射MPTP后10～14（12.0±1.67）天，臨床評(píng)分達(dá)到10(10.5±0.55)分，此時(shí)所有實(shí)驗(yàn)猴均逐漸出現(xiàn)典型的運(yùn)動(dòng)損害癥狀，包括運(yùn)動(dòng)遲緩、隨意運(yùn)動(dòng)活動(dòng)減少、屈曲體態(tài)、姿勢(shì)步態(tài)不穩(wěn)，肌強(qiáng)直、間斷性僵直發(fā)作及姿勢(shì)性或靜止性震顫。在其后約1周的時(shí)間（開始注射MPTP后18.8±2.9天）臨床癥狀持續(xù)進(jìn)展并進(jìn)入相對(duì)穩(wěn)定期，此時(shí)臨床評(píng)分為（19.5±4.8）分。 第二部分MPTP損傷對(duì)食蟹猴嗅球神經(jīng)遞質(zhì)表達(dá)的影響 方法 摘取模型組和對(duì)照組食蟹猴嗅球后制成石蠟切片。我們首先選擇的抗體針對(duì)的抗原物質(zhì)為單胺類神經(jīng)遞質(zhì)DA、5-HT。兩種抗體分別染模型組和對(duì)照組各25張石蠟切片，再?gòu)?5張食蟹猴嗅球石蠟切片中隨機(jī)選取5張，選取視野，顯微鏡攝片，分析免疫組化圖片，測(cè)平均光密度，計(jì)陽(yáng)性細(xì)胞數(shù)。 我們也選擇了氨基酸類神經(jīng)遞質(zhì)GABA和ChAT，研究方法同上。多巴胺系統(tǒng)與帕金森病頗具關(guān)聯(lián)，這里我們除了研究DA，還研究多巴胺通路上的信號(hào)分子受體和轉(zhuǎn)運(yùn)體蛋白，分別為DARPP32D1R D2R DAT。 結(jié)果 與對(duì)照組比較，MPTP組的單胺類神經(jīng)遞質(zhì)DA和5-HT的陽(yáng)性細(xì)胞數(shù)和陽(yáng)性纖維量均減少。 與對(duì)照組比較，MPTP組氨基酸類神經(jīng)遞質(zhì)GABA的陽(yáng)性細(xì)胞數(shù)和陽(yáng)性細(xì)胞量均減少。 與對(duì)照組比較，MPTP組ChAT陽(yáng)性細(xì)胞數(shù)與陽(yáng)性纖維量均減少。 與對(duì)照組比較，MPTP組DARPP32D1R D2R DAT陽(yáng)性細(xì)胞數(shù)與陽(yáng)性纖維量均減少，其中以DAT減少程度尤為顯著。 第三部分MPTP損傷對(duì)食蟹猴嗅球膠質(zhì)細(xì)胞的影響 方法 以抗Iba1抗體、抗GFAP抗體、抗TNF-α抗體用SABC法染色，每組從25張食蟹猴嗅球石蠟切片中隨機(jī)選取5張，顯微鏡攝片，分析免疫組化圖片，測(cè)平均光密度，計(jì)陽(yáng)性細(xì)胞數(shù)。從而觀察MPTP損傷對(duì)食蟹猴嗅球膠質(zhì)細(xì)胞的影響。 結(jié)果 與對(duì)照組比較，MPTP組Iba1陽(yáng)性物質(zhì)增多，提示小膠質(zhì)細(xì)胞有增殖。 與對(duì)照組比較，MPTP組GFAP陽(yáng)性物質(zhì)增多，提示星形膠質(zhì)細(xì)胞有增殖。 與對(duì)照組比較，MPTP組TNF-α陽(yáng)性物質(zhì)增多，提示有炎性反應(yīng)。 第四部分MPTP損傷對(duì)食蟹猴嗅球細(xì)胞凋亡的影響 方法 用抗Bcl2和抗caspase3抗體以SABC法染色，每組從25張食蟹猴嗅球石蠟切片中隨機(jī)選取5張，選取視野后顯微鏡攝片，分析免疫組化圖片，測(cè)平均光密度，計(jì)陽(yáng)性細(xì)胞數(shù)。觀察MPTP損傷對(duì)食蟹猴嗅球凋亡信號(hào)的影響。 結(jié)果 與對(duì)照組比較，MPTP組Bcl2陽(yáng)性物質(zhì)減少，caspase3陽(yáng)性物質(zhì)增多，提示細(xì)胞凋亡異常，呈上升。 結(jié)論 食蟹猴嗅球的組織結(jié)構(gòu)是環(huán)狀的多層結(jié)構(gòu)。 MPTP損傷造成了食蟹猴嗅球神經(jīng)遞質(zhì)表達(dá)的減少。 MPTP損傷造成了食蟹猴嗅球膠質(zhì)細(xì)胞的增生和炎癥反應(yīng)，免疫反應(yīng)。
[Abstract]:research objective
Parkinson's disease is more likely to be seen as a disease characterized by olfactory impairment than motor impairment, unlike traditional motor system cognition. The pathological changes of olfactory bulb, an important hub in olfactory pathway, are also extensively studied in this disease. MPTP non-human primate Parkinson's disease model is the most ideal study to date. Animal models of the pathogenesis of Parkinson's disease.
The aim of this study was to observe the neuroanatomical and neurochemical characteristics of the olfactory bulb in cynomolgus monkeys by immunohistochemical methods, including: 1. histological characteristics of the olfactory bulb in cynomolgus monkeys; 2. expression of major neurotransmitters and distribution of glial cells in the olfactory bulb of cynomolgus monkeys; 3. MPTP injury induced neurotransmitter expression in the olfactory bulb of cynomolgus, glial cells and cell apoptosis. The effects of MPTP injury on the expression of major neurotransmitters, apoptosis signals and glial cells in the olfactory bulb of cynomolgus monkeys can provide some background knowledge for the study of Parkinson's disease.
Part one establishment of MPTP cynomolgus Parkinson disease model
Method
Six healthy adult cynomolgus monkeys were raised, cared for and managed in animal centres in strict accordance with AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care) regulations and requirements. Fluid, slowly injected into the lower extremity veins, once a day in the morning (9AM). Individualized principles were adopted, with typical clinical symptoms as the end point of injection.
Result
From 10 to 14 days after MPTP injection, the clinical score reached 10 (10.5 (+ 0.55) points. At this time, all the monkeys gradually developed typical symptoms of motor impairment, including slow movement, decreased voluntary movement, flexion posture, instability of postural gait, muscular rigidity, intermittent stiffness attacks and postural or static tremors. (18.8 (+ 2.9) days after MPTP injection) the clinical symptoms progressed continuously and entered a relatively stable phase, with a clinical score of (19.5 (+ 4.8).
The second part is the effect of MPTP damage on the expression of neurotransmitters in the olfactory bulb of cynomolgus monkeys.
Method
Paraffin sections were made from the olfactory bulbs of the model and control groups. Firstly, the antigens targeted by the antibodies against DA and 5-HT were stained with 25 paraffin sections of the model group and 25 paraffin sections of the control group, and then 5 paraffin sections were randomly selected from 25 paraffin sections of the olfactory bulbs of the cynomolgus monkeys. Immunohistochemical analysis was used to measure the mean optical density and count the number of positive cells.
We also chose amino acid neurotransmitters GABA and ChAT, the same methodology as above. The dopamine system is associated with Parkinson's disease. Here we study not only DA, but also the receptors and transporters of the dopamine pathway, DARPP32D1R D2R DAT.
Result
Compared with the control group, the number of DA and 5-HT positive cells and the amount of positive fibers in MPTP group were decreased.
Compared with the control group, the number of positive neurons and the number of positive cells of amino acid neurotransmitters GABA in group MPTP were decreased.
Compared with the control group, the number of ChAT positive cells and the number of positive fibers in group MPTP decreased.
Compared with the control group, the number of DARPP32D1R D2R DAT positive cells and the amount of DAR DAT positive fibers in MPTP group were decreased, especially the decrease of DAT.
The third part is the effect of MPTP damage on the glial cells in the olfactory bulb of cynomolgus monkeys.
Method
Anti-Iba1 antibody, anti-GFAP antibody and anti-TNF-alpha antibody were stained with SABC method. Five paraffin sections of the olfactory bulb of cynomolgus monkeys were randomly selected from 25 paraffin sections in each group. Immunohistochemical images were analyzed. Mean optical density was measured and the number of positive cells was counted.
Result
Compared with control group, Iba1 positive substance increased in group MPTP, suggesting microglia proliferation.
Compared with the control group, GFAP positive substance increased in group MPTP, suggesting astrocyte proliferation.
Compared with the control group, there was an increase in TNF- alpha positive substance in group MPTP, suggesting an inflammatory response.
The fourth part is the effect of MPTP damage on the apoptosis of olfactory bulb cells in cynomolgus monkeys.
Method
Anti-Bcl-2 and anti-caspase-3 antibodies were stained with SABC method. Five paraffin sections of the olfactory bulb of cynomolgus monkeys were randomly selected from each group. Immunohistochemical images were taken under microscope after visual field. The average optical density was measured and the number of positive cells was counted. The effect of MPTP injury on the apoptotic signal of the olfactory bulb of cynomolgus monkeys was observed.
Result
Compared with the control group, the positive substance of Bcl 2 decreased and caspase 3 increased in MPTP group, suggesting that apoptosis was abnormal and increased.
conclusion
The structure of the olfactory bulb of cynomolgus monkeys is a circular multilayer structure.
MPTP damage reduced the expression of neurotransmitters in the olfactory bulb of cynomolgus monkeys.
MPTP damage caused the proliferation and inflammatory reaction of the cells in the olfactory bulb of cynomolgus monkeys.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R742.5
[Abstract]:research objective
Parkinson's disease is more likely to be seen as a disease characterized by olfactory impairment than motor impairment, unlike traditional motor system cognition. The pathological changes of olfactory bulb, an important hub in olfactory pathway, are also extensively studied in this disease. MPTP non-human primate Parkinson's disease model is the most ideal study to date. Animal models of the pathogenesis of Parkinson's disease.
The aim of this study was to observe the neuroanatomical and neurochemical characteristics of the olfactory bulb in cynomolgus monkeys by immunohistochemical methods, including: 1. histological characteristics of the olfactory bulb in cynomolgus monkeys; 2. expression of major neurotransmitters and distribution of glial cells in the olfactory bulb of cynomolgus monkeys; 3. MPTP injury induced neurotransmitter expression in the olfactory bulb of cynomolgus, glial cells and cell apoptosis. The effects of MPTP injury on the expression of major neurotransmitters, apoptosis signals and glial cells in the olfactory bulb of cynomolgus monkeys can provide some background knowledge for the study of Parkinson's disease.
Part one establishment of MPTP cynomolgus Parkinson disease model
Method
Six healthy adult cynomolgus monkeys were raised, cared for and managed in animal centres in strict accordance with AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care) regulations and requirements. Fluid, slowly injected into the lower extremity veins, once a day in the morning (9AM). Individualized principles were adopted, with typical clinical symptoms as the end point of injection.
Result
From 10 to 14 days after MPTP injection, the clinical score reached 10 (10.5 (+ 0.55) points. At this time, all the monkeys gradually developed typical symptoms of motor impairment, including slow movement, decreased voluntary movement, flexion posture, instability of postural gait, muscular rigidity, intermittent stiffness attacks and postural or static tremors. (18.8 (+ 2.9) days after MPTP injection) the clinical symptoms progressed continuously and entered a relatively stable phase, with a clinical score of (19.5 (+ 4.8).
The second part is the effect of MPTP damage on the expression of neurotransmitters in the olfactory bulb of cynomolgus monkeys.
Method
Paraffin sections were made from the olfactory bulbs of the model and control groups. Firstly, the antigens targeted by the antibodies against DA and 5-HT were stained with 25 paraffin sections of the model group and 25 paraffin sections of the control group, and then 5 paraffin sections were randomly selected from 25 paraffin sections of the olfactory bulbs of the cynomolgus monkeys. Immunohistochemical analysis was used to measure the mean optical density and count the number of positive cells.
We also chose amino acid neurotransmitters GABA and ChAT, the same methodology as above. The dopamine system is associated with Parkinson's disease. Here we study not only DA, but also the receptors and transporters of the dopamine pathway, DARPP32D1R D2R DAT.
Result
Compared with the control group, the number of DA and 5-HT positive cells and the amount of positive fibers in MPTP group were decreased.
Compared with the control group, the number of positive neurons and the number of positive cells of amino acid neurotransmitters GABA in group MPTP were decreased.
Compared with the control group, the number of ChAT positive cells and the number of positive fibers in group MPTP decreased.
Compared with the control group, the number of DARPP32D1R D2R DAT positive cells and the amount of DAR DAT positive fibers in MPTP group were decreased, especially the decrease of DAT.
The third part is the effect of MPTP damage on the glial cells in the olfactory bulb of cynomolgus monkeys.
Method
Anti-Iba1 antibody, anti-GFAP antibody and anti-TNF-alpha antibody were stained with SABC method. Five paraffin sections of the olfactory bulb of cynomolgus monkeys were randomly selected from 25 paraffin sections in each group. Immunohistochemical images were analyzed. Mean optical density was measured and the number of positive cells was counted.
Result
Compared with control group, Iba1 positive substance increased in group MPTP, suggesting microglia proliferation.
Compared with the control group, GFAP positive substance increased in group MPTP, suggesting astrocyte proliferation.
Compared with the control group, there was an increase in TNF- alpha positive substance in group MPTP, suggesting an inflammatory response.
The fourth part is the effect of MPTP damage on the apoptosis of olfactory bulb cells in cynomolgus monkeys.
Method
Anti-Bcl-2 and anti-caspase-3 antibodies were stained with SABC method. Five paraffin sections of the olfactory bulb of cynomolgus monkeys were randomly selected from each group. Immunohistochemical images were taken under microscope after visual field. The average optical density was measured and the number of positive cells was counted. The effect of MPTP injury on the apoptotic signal of the olfactory bulb of cynomolgus monkeys was observed.
Result
Compared with the control group, the positive substance of Bcl 2 decreased and caspase 3 increased in MPTP group, suggesting that apoptosis was abnormal and increased.
conclusion
The structure of the olfactory bulb of cynomolgus monkeys is a circular multilayer structure.
MPTP damage reduced the expression of neurotransmitters in the olfactory bulb of cynomolgus monkeys.
MPTP damage caused the proliferation and inflammatory reaction of the cells in the olfactory bulb of cynomolgus monkeys.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R742.5
【共引文獻(xiàn)】
相關(guān)期刊論文 前10條
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6 葉芳;易剛;肖軍;;中國(guó)人群帕金森病危險(xiǎn)因素及保護(hù)性因素的Meta分析[J];臨床神經(jīng)病學(xué)雜志;2014年02期
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