氧糖剥夺后海马神经元中AP4M1表达变化及其对AMPA受体运输的调控作用
发布时间：2018-08-13 21:09
【摘要】：目的研究已证实衔接蛋白复合物-4(adaptor protein complex-4, AP-4)基因突变与家族性隐性遗传性脑瘫综合征相关,且其μ亚基基因(adaptor-related protein complex 4, mu 1 subunit, AP4M1)的突变可引起脑发育缺陷而导致先天性痉挛性瘫痪(congenital spastic tetraplegia, CST)并模拟了谷氨酸介导的围产期脑白质损伤,可能的机制为AP4M1介导的APMA受体的异常分布引起的。然而AP4M1在缺氧性脑损伤中的表达变化及可能通过何种途径产生作用尚不明确,本课题的目的是研究AP4M1在损伤的神经元中表达量和分布的变化,以及AP4介导AMPA受体运输的分子机制。以期探寻缺氧缺血性脑损伤的新机制和防治的新策略。材料及方-法本课题以原代培养的胎鼠海马神经元为实验材料,采用氧糖剥夺建立缺氧性脑损伤的细胞模型,免疫荧光标记神经元特异性烯醇化酶(neuron specific enalose, NSE)以确定培养细胞中神经元的纯度。实验分为正常对照组及氧糖剥夺组,Real-time PCR、western blot及免疫比色分析检测的时间点为氧糖剥夺后0、12以及24小时,细胞免疫荧光双染色及免疫共沉淀检测的时间点为氧糖剥夺后0小时。分别采用Real-time PCR及western blot方法检测AP4M1和AMPARs的mRNA及蛋白表达量的变化。进行细胞免疫荧光双染色标记AP4M1与MAP2、AP4M1 与 Tau-1、G1uR1-4与MAP2来研究AP4M1及AMPA受体(AMPA receptors, AMPARs)在神经元内树突／轴突的分布。采用免疫比色分析标记表面及总的AP4M1、G1uR1及GluR2来检测其在神经元膜表面的分布。进行细胞免疫荧光双染色标记AP4M1与G1uR1、G1uR2 及 G1uR4,检测AP4M1与AMPARs在氧糖剥夺前后的共分布情况。将AP4M1分别与GluR1及GluR2进行免疫共沉淀来探讨其相互之间是否存在直接结合作用。结果1.海马神经元的原代培养、鉴定及氧糖剥夺细胞模型的建立：原代培养的胎鼠海马神经元生长良好,在种植后第13天发育成熟,经细胞免疫荧光染色鉴定神经元在所有培养细胞中的纯度超过95%。氧糖剥夺后24小时,超过90%的神经元已经死亡,部分表现为凋亡,部分表现为坏死。2. Real-time PCR检测：氧糖剥夺组AP4M1 mRNA表达量较正常对照组明显下调。氧糖剥夺后0小时AP4M1 mRNA表达量大约是正常对照组的77.9%(p=0.009, n=8),即氧糖剥夺引起表达下降1.28倍；然后AP4M1 mRNA表达量在氧糖剥夺后12小时急剧下降至正常对照组的34.8%(p0.001,n=8),氧糖剥夺后24小时下降至34.2%(p0.001,n=8)。正常对照组不同时间点之间G1uR1-4 mRNA表达量没有明显统计学差异；氧糖剥夺后0小时GluR1-4mRNA表达量与正常对照组之间也没有明显统计学差异；氧糖剥夺后12小时G1uR1-3 mRNA表达量与正常对照组之间没有明显统计学差异,G1uR4 mRNA 表达量较正常对照组轻度下降,为正常对照组的71.8%(p=0.010,n=8)；氧糖剥夺后24小时GluR1 mRNA和GluR2 mRNA表达量较正常对照组下降,分别为正常对照组的20.2%(p0.001,n=8)和20.8%(p0.001,n=8),G1uR4 mRNA仍表现为轻度下降,为正常对照组的62.0%(p=0.009,n=8),G1uR3 mRNA 表达量仍与正常对照组之间没有统计学差异。3. western blot检测：氧糖剥夺后0小时AP4M1蛋白表达量与正常对照组之间没有明显统计学差异(p=0.835,n=8),表达下调在氧糖剥夺后12小时可以检测到,蛋白表达量约为正常对照组的40.87%(p0.001,n=8)。这一下调作用在OGD后24小时更加明显,AP4M1蛋白表达量仅为正常对照组的19.79%(p0.001,n=8)。氧糖剥夺组GluR1-4蛋白表达量在氧糖剥夺后0小时、12小时及24小时均与正常对照组没有明显统计学差异。4.免疫荧光染色分析：AP4M1在正常对照组神经元中主要表达于树突,而氧糖剥夺后0小时即可观察到其再分布至神经元的轴突部分；GluR2在正常对照照组神经元中表达于树突,氧糖剥夺后在树突部分表达减少；而GluRl及GluR4在正常对照组神经元中主要表达于胞体部分,少量表达于树突,在氧糖剥夺后分布变化不明显。5.免疫比色分析：OGD后0小时及12小时氧糖剥夺组和正常对照组神经元的AP4M1总表达量之间没有明显统计学差异；OGD后24小时,氧糖剥夺组神经元的AP4M1总表达量下降(p0.001,n=6),约为正常对照组的79.8%。OGD后0小时AP4M1在氧糖剥夺组和正常对照组神经元的细胞表面表达量及表面表达率没有明显统计学差异；OGD后12小时AP4M1在神经元的表面表达量下降(p0.001,n=6),约为正常对照组的62.8%；OGD后24小时AP4M1在神经元的表面表达量继续下降至正常对照组的37.3%(p0.001,n=6)。OGD后12小时AP4M1在神经元的表面表达率下降(p0.001,n=6),约为正常对照组的58.7%；OGD后24小时AP4M1在神经元的表面表达率继续下降至正常对照组的46.6%(p0.001,n=6)。氧糖剥夺后0小时、12小时及24小时,氧糖剥夺组和正常对照组神经元的GluRl的总表达量、表面表达量及表面表达率均没有明显统计学差异。OGD后0小时、12小时及24小时,氧糖剥夺组和正常对照组神经元的GluR2总表达量之间没有明显统计学差异。OGD后0小时GluR2在氧糖剥夺组和正常对照组神经元的细胞表面表达量及表面表达率均没有明显统计学差异；OGD后12小时GluR2在神经元的表面表达量下降(p0.001,n=6),约为正常对照组的61.9%；OGD后24小时,GluR2在神经元的表面表达量继续下降至正常对照组的28.4%(p0.001,n=6)。OGD后12小时,GluR2在神经元的表面表达率下降(p0.001,n=6),约为正常对照组的61.2%；OGD后24小时,GluR2在神经元的表面表达率继续下降至正常对照组的29.2%(p0.001,n=6)。6.免疫荧光双染色分析：GluRl及GluR4与AP4M1在正常对照组及氧糖剥夺组神经元中融合度较低, 而GluR2与AP4M1在正常对照组及氧糖剥夺组神经元中融合度均较高,分布一致。7.免疫共沉淀分析：在『F常对照组和氧糖剥夺组神经元中,未检测到GluRl与AP4M1之间有直接结合作用,GluR2与AP4M1之间也没有检测到直接结合作用。结论在氧糖剥夺后的海马神经元中不仅出现AP4MI的mRNA和蛋白表达明显下调,也出现AP4M1从树突再分布至轴突的分布异常,而且在神经元细胞膜表面的分布减少；氧糖剥夺后海马神经元中的G1uR2 mRNA表达下调,且GIuR2蛋白在海马神经元中的分布发生变化,在树突和细胞表面分布减少；AP4M1可能间接调控氧糖剥夺后的海马神经元中的GIuR2转运。
[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.
【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R743
本文编号：2182198
[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.
【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R743
【参考文献】
相关期刊论文 前1条
1 新生儿缺氧缺血性脑病治疗协作组;新生儿缺氧缺血性脑病多中心治疗的报告[J];中国实用儿科杂志;2000年06期
，本文编号：2182198
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