【摘要】：目的研究已證實銜接蛋白復合物-4(adaptor protein complex-4, AP-4)基因突變與家族性隱性遺傳性腦癱綜合征相關,且其μ亞基基因(adaptor-related protein complex 4, mu 1 subunit, AP4M1)的突變可引起腦發(fā)育缺陷而導致先天性痙攣性癱瘓(congenital spastic tetraplegia, CST)并模擬了谷氨酸介導的圍產期腦白質損傷,可能的機制為AP4M1介導的APMA受體的異常分布引起的。然而AP4M1在缺氧性腦損傷中的表達變化及可能通過何種途徑產生作用尚不明確,本課題的目的是研究AP4M1在損傷的神經(jīng)元中表達量和分布的變化,以及AP4介導AMPA受體運輸?shù)姆肿訖C制。以期探尋缺氧缺血性腦損傷的新機制和防治的新策略。材料及方-法本課題以原代培養(yǎng)的胎鼠海馬神經(jīng)元為實驗材料,采用氧糖剝奪建立缺氧性腦損傷的細胞模型,免疫熒光標記神經(jīng)元特異性烯醇化酶(neuron specific enalose, NSE)以確定培養(yǎng)細胞中神經(jīng)元的純度。實驗分為正常對照組及氧糖剝奪組,Real-time PCR、western blot及免疫比色分析檢測的時間點為氧糖剝奪后0、12以及24小時,細胞免疫熒光雙染色及免疫共沉淀檢測的時間點為氧糖剝奪后0小時。分別采用Real-time PCR及western blot方法檢測AP4M1和AMPARs的mRNA及蛋白表達量的變化。進行細胞免疫熒光雙染色標記AP4M1與MAP2、AP4M1 與 Tau-1、G1uR1-4與MAP2來研究AP4M1及AMPA受體(AMPA receptors, AMPARs)在神經(jīng)元內樹突／軸突的分布。采用免疫比色分析標記表面及總的AP4M1、G1uR1及GluR2來檢測其在神經(jīng)元膜表面的分布。進行細胞免疫熒光雙染色標記AP4M1與G1uR1、G1uR2 及 G1uR4,檢測AP4M1與AMPARs在氧糖剝奪前后的共分布情況。將AP4M1分別與GluR1及GluR2進行免疫共沉淀來探討其相互之間是否存在直接結合作用。結果1.海馬神經(jīng)元的原代培養(yǎng)、鑒定及氧糖剝奪細胞模型的建立：原代培養(yǎng)的胎鼠海馬神經(jīng)元生長良好,在種植后第13天發(fā)育成熟,經(jīng)細胞免疫熒光染色鑒定神經(jīng)元在所有培養(yǎng)細胞中的純度超過95%。氧糖剝奪后24小時,超過90%的神經(jīng)元已經(jīng)死亡,部分表現(xiàn)為凋亡,部分表現(xiàn)為壞死。2. Real-time PCR檢測：氧糖剝奪組AP4M1 mRNA表達量較正常對照組明顯下調。氧糖剝奪后0小時AP4M1 mRNA表達量大約是正常對照組的77.9%(p=0.009, n=8),即氧糖剝奪引起表達下降1.28倍；然后AP4M1 mRNA表達量在氧糖剝奪后12小時急劇下降至正常對照組的34.8%(p0.001,n=8),氧糖剝奪后24小時下降至34.2%(p0.001,n=8)。正常對照組不同時間點之間G1uR1-4 mRNA表達量沒有明顯統(tǒng)計學差異；氧糖剝奪后0小時GluR1-4mRNA表達量與正常對照組之間也沒有明顯統(tǒng)計學差異；氧糖剝奪后12小時G1uR1-3 mRNA表達量與正常對照組之間沒有明顯統(tǒng)計學差異,G1uR4 mRNA 表達量較正常對照組輕度下降,為正常對照組的71.8%(p=0.010,n=8)；氧糖剝奪后24小時GluR1 mRNA和GluR2 mRNA表達量較正常對照組下降,分別為正常對照組的20.2%(p0.001,n=8)和20.8%(p0.001,n=8),G1uR4 mRNA仍表現(xiàn)為輕度下降,為正常對照組的62.0%(p=0.009,n=8),G1uR3 mRNA 表達量仍與正常對照組之間沒有統(tǒng)計學差異。3. western blot檢測：氧糖剝奪后0小時AP4M1蛋白表達量與正常對照組之間沒有明顯統(tǒng)計學差異(p=0.835,n=8),表達下調在氧糖剝奪后12小時可以檢測到,蛋白表達量約為正常對照組的40.87%(p0.001,n=8)。這一下調作用在OGD后24小時更加明顯,AP4M1蛋白表達量僅為正常對照組的19.79%(p0.001,n=8)。氧糖剝奪組GluR1-4蛋白表達量在氧糖剝奪后0小時、12小時及24小時均與正常對照組沒有明顯統(tǒng)計學差異。4.免疫熒光染色分析：AP4M1在正常對照組神經(jīng)元中主要表達于樹突,而氧糖剝奪后0小時即可觀察到其再分布至神經(jīng)元的軸突部分；GluR2在正常對照照組神經(jīng)元中表達于樹突,氧糖剝奪后在樹突部分表達減少；而GluRl及GluR4在正常對照組神經(jīng)元中主要表達于胞體部分,少量表達于樹突,在氧糖剝奪后分布變化不明顯。5.免疫比色分析：OGD后0小時及12小時氧糖剝奪組和正常對照組神經(jīng)元的AP4M1總表達量之間沒有明顯統(tǒng)計學差異；OGD后24小時,氧糖剝奪組神經(jīng)元的AP4M1總表達量下降(p0.001,n=6),約為正常對照組的79.8%。OGD后0小時AP4M1在氧糖剝奪組和正常對照組神經(jīng)元的細胞表面表達量及表面表達率沒有明顯統(tǒng)計學差異；OGD后12小時AP4M1在神經(jīng)元的表面表達量下降(p0.001,n=6),約為正常對照組的62.8%；OGD后24小時AP4M1在神經(jīng)元的表面表達量繼續(xù)下降至正常對照組的37.3%(p0.001,n=6)。OGD后12小時AP4M1在神經(jīng)元的表面表達率下降(p0.001,n=6),約為正常對照組的58.7%；OGD后24小時AP4M1在神經(jīng)元的表面表達率繼續(xù)下降至正常對照組的46.6%(p0.001,n=6)。氧糖剝奪后0小時、12小時及24小時,氧糖剝奪組和正常對照組神經(jīng)元的GluRl的總表達量、表面表達量及表面表達率均沒有明顯統(tǒng)計學差異。OGD后0小時、12小時及24小時,氧糖剝奪組和正常對照組神經(jīng)元的GluR2總表達量之間沒有明顯統(tǒng)計學差異。OGD后0小時GluR2在氧糖剝奪組和正常對照組神經(jīng)元的細胞表面表達量及表面表達率均沒有明顯統(tǒng)計學差異；OGD后12小時GluR2在神經(jīng)元的表面表達量下降(p0.001,n=6),約為正常對照組的61.9%；OGD后24小時,GluR2在神經(jīng)元的表面表達量繼續(xù)下降至正常對照組的28.4%(p0.001,n=6)。OGD后12小時,GluR2在神經(jīng)元的表面表達率下降(p0.001,n=6),約為正常對照組的61.2%；OGD后24小時,GluR2在神經(jīng)元的表面表達率繼續(xù)下降至正常對照組的29.2%(p0.001,n=6)。6.免疫熒光雙染色分析：GluRl及GluR4與AP4M1在正常對照組及氧糖剝奪組神經(jīng)元中融合度較低, 而GluR2與AP4M1在正常對照組及氧糖剝奪組神經(jīng)元中融合度均較高,分布一致。7.免疫共沉淀分析：在『F常對照組和氧糖剝奪組神經(jīng)元中,未檢測到GluRl與AP4M1之間有直接結合作用,GluR2與AP4M1之間也沒有檢測到直接結合作用。結論在氧糖剝奪后的海馬神經(jīng)元中不僅出現(xiàn)AP4MI的mRNA和蛋白表達明顯下調,也出現(xiàn)AP4M1從樹突再分布至軸突的分布異常,而且在神經(jīng)元細胞膜表面的分布減少；氧糖剝奪后海馬神經(jīng)元中的G1uR2 mRNA表達下調,且GIuR2蛋白在海馬神經(jīng)元中的分布發(fā)生變化,在樹突和細胞表面分布減少；AP4M1可能間接調控氧糖剝奪后的海馬神經(jīng)元中的GIuR2轉運。
[Abstract]:Objective To study the relationship between the mutation of adaptor protein complex-4 (AP-4) gene and familial recessive cerebral palsy syndrome, and the mutation of its Mu subunit (AP4M1) gene can lead to brain development defects and congenital spastic paralysis. Teraplegia (CST) also mimics glutamate-mediated perinatal white matter injury, possibly caused by the abnormal distribution of APMA receptors mediated by AP4M1. However, the expression of AP4M1 in hypoxic brain injury and the possible pathway through which AP4M1 may play a role are still unclear. The changes of expression and distribution of AMPA receptor and the molecular mechanism of AP4 mediating AMPA receptor transport were studied in order to explore the new mechanism of hypoxic-ischemic brain damage and new strategies for prevention and treatment. Neuron specific enolase (NSE) was recorded to determine the purity of cultured neurons. The experiment was divided into normal control group and oxygen-glucose deprivation group. Real-time PCR, Western blot and immunochromatographic analysis were performed at 0, 12 and 24 hours after oxygen-glucose deprivation. The expression of AP4M1 and AMPARs was detected by Real-time PCR and Western blot respectively. The dendrites of AP4M1 and AMPARs in neurons were studied by immunofluorescence double staining of AP4M1 and MAP2, AP4M1 and Tau-1, G1uR1-4 and MAP2. Distribution of AP4M1, G1uR1 and GluR2 on the surface of neuron membrane was detected by immunocolorimetric analysis. AP4M1 and G1uR1, G1uR2 and G1uR4 were labeled by immunofluorescence double staining. The distribution of AP4M1 and AMPARs before and after glucose deprivation was detected. AP4M1 was immunized with GluR1 and GluR2 respectively. Results 1. Primary culture and identification of hippocampal neurons and establishment of oxygen-glucose deprivation cell model: The primary cultured hippocampal neurons grew well and matured on the 13th day after implantation. The neurons were identified by immunofluorescence staining in all cultured cells. The purity of AP4M1 was over 95%. More than 90% of the neurons died 24 hours after glucose and oxygen deprivation, some of them were apoptotic and some were necrotic. 2. Real-time PCR showed that the expression of AP4M1 mRNA in the glucose and oxygen deprivation group was significantly lower than that in the normal control group. The expression of AP4M1 mRNA was about 77.9% (p = 0.009, n = 8) in the glucose and oxygen deprivation group 0 hours after glucose deprivation. The expression of AP4M1 mRNA decreased by 1.28 fold after oxygen-glucose deprivation, and then decreased sharply to 34.8% (p0.001, n=8) at 12 hours after oxygen-glucose deprivation and 34.2% (p0.001, n=8) at 24 hours after oxygen-glucose deprivation. GluR1-4 mRNA expression was not significantly different between the normal control group and the oxygen-glucose deprivation group; G1uR1-3 mRNA expression 12 hours after oxygen-glucose deprivation was not significantly different from the normal control group, G1uR4 mRNA expression was slightly lower than the normal control group, 71.8% of the normal control group (p = 0.010, n = 8); GluR1 m 24 hours after oxygen-glucose deprivation. The expression levels of RNA and GluR2 mRNA in normal control group were 20.2% (p0.001, n = 8) and 20.8% (p0.001, n = 8), respectively. G1uR4 mRNA was still slightly decreased, 62.0% (p = 0.009, n = 8) in normal control group, and there was no significant difference between G1uR3 mRNA and normal control group. There was no significant difference in the expression of AP4M1 protein between the two groups (p = 0.835, n = 8). The down-regulation of AP4M1 protein was detected 12 hours after glucose and oxygen deprivation. The down-regulation was about 40.87% (p0.001, n = 8) of the normal control group. The down-regulation was more obvious 24 hours after OGD, and the expression of AP4M1 protein was only 19.7% of the normal control group. 9% (p0.001, n = 8). The expression of GluR1-4 protein in oxygen-glucose deprivation group was not significantly different from that in normal control group at 0, 12 and 24 hours after oxygen-glucose deprivation. GluR2 was expressed in dendrites in normal control neurons and decreased in dendrites after oxygen and glucose deprivation, while GluRl and GluR4 were mainly expressed in somatic neurons and a small amount in dendrites in normal control neurons. 5. Immunocolorimetric analysis: 0 and 12 hours after OGD There was no significant difference in the total expression of AP4M1 between the oxygen-glucose deprivation group and the normal control group. The total expression of AP4M1 in the oxygen-glucose deprivation group decreased 24 hours after OGD (p0.001, n=6), about 79.8% of that in the normal control group. The expression of AP4M1 on the surface of neurons decreased 12 hours after OGD (p 0.001, n = 6), which was about 62.8% of that in the normal control group. The expression of AP4M1 on the surface of neurons continued to decrease to 37.3% of that in the normal control group 24 hours after OGD (p 0.001, n = 6). The expression of AP4M1 on the surface of neurons decreased 12 hours after OGD (p 0.001, n = 6). The expression of AP4M1 on the neuron surface decreased to 46.6% (p0.001, n = 6) 24 hours after OGD. The total expression of GluRl, the surface expression and the surface expression rate of GluRl in the neurons of the oxygen-glucose deprivation group and the normal control group were not clear at 0, 12 and 24 hours after oxygen-glucose deprivation. There was no significant difference in the total expression of GluR2 between the oxygen-glucose deprivation group and the normal control group at 0, 12 and 24 hours after OGD. The expression of GluR2 on the surface of neurons decreased (p0.001, n = 6), about 61.9% of that in the normal control group; the expression of GluR2 on the surface of neurons continued to decrease to 28.4% of that in the normal control group 24 hours after OGD (p0.001, n = 6). 12 hours after OGD, the expression of GluR2 on the surface of neurons decreased (p0.001, n = 6), about 61.2% of that in the normal control group; and 24 hours after OGD, the expression of GluR2 on the surface of neurons decreased (p0.001, n Immunofluorescence double staining analysis showed that GluRl, GluR4 and AP4M1 fused poorly in the neurons of normal control group and oxygen-glucose deprivation group, while GluR2 and AP4M1 fused well in the neurons of normal control group and oxygen-glucose deprivation group. Immunocoprecipitation analysis: No direct binding between GluRl and AP4M1 was detected in the neurons of F normal control group and oxygen-glucose deprivation group, and no direct binding between GluR2 and AP4M1 was detected. AP4M1 redistributes abnormally from dendrites to axons and decreases on the surface of cell membrane; G1uR2 mRNA expression is down-regulated in hippocampal neurons after oxygen-glucose deprivation, and GIuR2 protein distribution changes in hippocampal neurons, and decreases in dendrites and cell surface distribution; AP4M1 may indirectly regulate oxygen-glucose deprivation. GIuR2 transport in hippocampal neurons.
【學位授予單位】：鄭州大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R743

【參考文獻】

相關期刊論文 前1條

1 新生兒缺氧缺血性腦病治療協(xié)作組;新生兒缺氧缺血性腦病多中心治療的報告[J];中國實用兒科雜志;2000年06期

，

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