阿尔茨海默病5xFAD转基因小鼠眼部病变研究

发布时间：2018-03-02 08:42

  本文关键词： 衰老 淀粉样前体蛋白 tau蛋白 磷酸化 5xFAD小鼠　出处：《中国人民解放军医学院》2013年博士论文　论文类型：学位论文

【摘要】：目的 1.研究淀粉样前体蛋白在5xFAD转基因小鼠眼球不同结构的蛋白分布。比较淀粉样前体蛋白、磷酸化淀粉样前体蛋白、β蛋白裂解酶、γ蛋白裂解酶在不同年龄组5xFAD转基因小鼠和B6SJL非转基因小鼠眼球不同结构中蛋白含量的变化。观察视紫红质在年龄、基因等不同因素作用下在5xFAD转基因小鼠和B6SJL非转基因小鼠视网膜的变化。 2．探索淀粉样前体蛋白、磷酸化淀粉样前体蛋白、β淀粉裂解酶、γ淀粉裂解酶和视紫红质在5xFAD转基因小鼠视网膜组织细胞亚结构的分布。 3.研究总tau蛋白和磷酸化tau蛋白在5xFAD转基因小鼠眼球不同结构的蛋白分布。检测不同位点磷酸化tau蛋白在5xFAD转基因小鼠和B6SJL非转基因小鼠眼球的表达。比较总tau蛋白和磷酸化tau蛋白在不同年龄组5xFAD转基因小鼠和B6SJL非转基因小鼠眼球蛋白表达的改变。 4.检测GSK3β和P-GSK3β含量在不同年龄组5xFAD转基因小鼠和B6SJL非转基因小鼠眼球蛋白表达的改变，探讨GSK3β通路是否参与磷酸化tau蛋白在5xFAD转基因小鼠眼部病变的过程。 方法 1.选取2月龄5xFAD转基因小鼠，用Western Blot观察淀粉样前体蛋白在角膜、晶状体、视网膜、视神经和大脑组织中的表达。再选取不同年龄组5xFAD转基因小鼠（2月和9月龄）和B6SJL非转基因小鼠（1周、1月、2月和9月龄），WesternBlot观察淀粉样前体蛋白、磷酸化淀粉样前体蛋白、β蛋白裂解酶、γ蛋白裂解酶和视紫红质在小鼠视网膜、视神经和大脑组织蛋白表达的改变。 2.选取9月龄5xFAD转基因小鼠，用组织亚细胞结构分层技术探测淀粉样前体蛋白、磷酸化淀粉样前体蛋白、β蛋白裂解酶、γ蛋白裂解酶和视紫红质在不同亚细胞结构如内质网、高尔基体和早期内涵体的分布。 3.选取2月龄5xFAD转基因小鼠，用Western Blot观察总tau蛋白和磷酸化tau蛋白在角膜、晶状体、视网膜、视神经和大脑组织中的表达。再选取不同年龄组5xFAD转基因小鼠（2月和9月龄）和（B6SJL非转基因小鼠1周、1月、2月和9月龄），蛋白纯化并降磷酸化处理后，Western Blot观察不同位点磷酸化tau蛋白在小鼠视网膜和大脑表达的异同，以及总tau蛋白和磷酸化tau蛋白在小鼠视网膜、视神经和大脑组织上蛋白表达的改变。 4.选取不同年龄组（2月和9月龄）5xFAD转基因小鼠和B6SJL非转基因小鼠，Western Blot观察GSK3β，P-GSK3β与磷酸化tau蛋白在视网膜和大脑组织上蛋白表达变化的关系。 结果 1.淀粉样前体蛋白在5xFAD转基因小鼠的角膜、晶状体、视网膜、视神经上均有分布，但表达形式不同，其中表达于视网膜和视神经上的蛋白形态最接近于大脑。发育期(1周龄至1月龄)，淀粉样前体蛋白含量在B6SJL非转基因小鼠视网膜上逐渐增加，且蛋白形式趋于成熟。成年期(2月龄至9月龄)，淀粉样前体蛋白含量在B6SJL非转基因小鼠和5xFAD转基因小鼠视网膜上均显著升高(p=0.02, p0.01)。2月龄时，5xFAD转基因小鼠视网膜淀粉样前体蛋白含量与B6SJL非转基因小鼠无明显差异，但9月龄时，5xFAD转基因小鼠视网膜淀粉样前体蛋白含量显著高于B6SJL非转基因小鼠(p0.01)。淀粉样前体蛋白含量在B6SJL非转基因小鼠和2月龄5xFAD转基因小鼠视神经上均较低，在9月龄5xFAD转基因小鼠显著升高(p0.01)。视网膜淀粉样前体蛋白含量变化与大脑表现一致。 2.发育期(1周龄至1月龄)，磷酸化淀粉样前体蛋白含量在B6SJL非转基因小鼠视网膜上较低。成年期(2月龄至9月龄)，磷酸化淀粉样前体蛋白含量在B6SJL非转基因小鼠和5xFAD转基因小鼠视网膜上均显著升高(p=0.03, p0.01)。且2月龄和9月龄时，5xFAD转基因小鼠视网膜磷酸化淀粉样前体蛋白含量均显著高于B6SJL非转基因小鼠(p0.01, p0.01)。磷酸化淀粉样前体蛋白含量在B6SJL非转基因小鼠和2月龄5xFAD转基因小鼠视神经上均较低，在9月龄5xFAD转基因小鼠显著升高(p0.01)。视网膜磷酸化淀粉样前体蛋白含量变化与大脑表现一致。 3.发育期(1周龄至1月龄)，视紫红质含量在B6SJL非转基因小鼠视网膜显著增加。成年期(2月龄至9月龄)，视紫红质含量在B6SJL非转基因小鼠视网膜有所下降，但在5xFAD转基因小鼠视网膜下降显著(p=0.01）。2月龄时，5xFAD转基因小鼠视网膜视紫红质含量与B6SJL非转基因小鼠无明显差异，但9月龄时，5xFAD转基因小鼠视网膜视紫红质含量显著低于B6SJL非转基因小鼠(p=0.01）。 4.发育期(1周龄至1月龄)，β蛋白裂解酶和γ淀粉裂解酶含量在B6SJL非转基因小鼠视网膜上显著降低。成年期(2月龄至9月龄)，β蛋白裂解酶和γ淀粉裂解酶含量在B6SJL非转基因小鼠和5xFAD转基因小鼠视网膜上均显著升高（p0.01,p=0.01）。且2月龄和9月龄时，5xFAD转基因小鼠视网膜β蛋白裂解酶和γ淀粉裂解酶含量均显著高于B6SJL非转基因小鼠（p0.01, p0.01）。视网膜β蛋白裂解酶和γ淀粉裂解酶含量变化与大脑表现一致。 5.9月龄5xFAD转基因小鼠视网膜组织亚细胞结构定位显示淀粉样前体蛋白位于内质网、高尔基体和早期内涵体，磷酸化淀粉样蛋白位于高尔基体，β蛋白裂解酶位于内质网和早期内涵体，，γ蛋白裂解酶位于内质网和高尔基体，而视紫红质位于内质网。 6.总tau蛋白和磷酸化tau蛋白仅在5xFAD转基因小鼠的视网膜和视神经上有分布，与大脑表现一致。在5xFAD转基因小鼠和B6SJL非转基因小鼠视网膜上主要表现为tau蛋白Ser396磷酸化位点(PHF-1)，与大脑表现一致。Ser202位点仅表现在9月龄5xFAD转基因小鼠视网膜且不确定，而Ser199/Thr205磷酸化位点在视网膜上则未检测到阳性表现。 7.发育期(1周龄至1月龄)，总tau蛋白含量在B6SJL非转基因小鼠视网膜上显著增加。成年期(2月龄至9月龄)，总tau蛋白含量在B6SJL非转基因小鼠和5xFAD转基因小鼠视网膜上均显著升高(p0.01, p0.01)。2月龄和9月龄时，5xFAD转基因小鼠视网膜总tau蛋白含量均显著高于B6SJL非转基因小鼠(p0.01, p0.01)。总tau蛋白含量在B6SJL非转基因小鼠和2月龄5xFAD转基因小鼠视神经上均较低，在9月龄5xFAD转基因小鼠显著升高。视网膜总tau蛋白含量变化与大脑表现一致。 8.发育期(1周龄至1月龄)，PHF-1含量和PHF-1与总tau蛋白含量的比值在B6SJL非转基因小鼠视网膜上显著较低。成年期(2月龄至9月龄)，PHF-1含量和PHF-1与总tau蛋白含量的比值在B6SJL非转基因小鼠视网膜上呈增高趋势，在转基因5xFAD小鼠视网膜上呈显著增高(p0.01, p0.01)。且2月龄时，5xFAD转基因小鼠视网膜PHF-1含量和PHF-1与总tau蛋白含量的比值与B6SJL非转基因小鼠无差异，但9月龄时，5xFAD转基因小鼠视网膜PHF-1含量和PHF-1与总tau蛋白含量的比值均显著高于B6SJL非转基因小鼠(p0.01, p0.01)。视网膜PHF-1含量变化与大脑表现一致。 9.成年期(2月龄至9月龄)，GSK3β含量在9月龄5xFAD转基因小鼠视网膜较同龄B6SJL非转基因小鼠和2月龄5xFAD转基因小鼠显著升高(p=0.02)，而P-GSK3β含量在9月龄5xFAD转基因小鼠视网膜较同龄B6SJL非转基因小鼠和2月龄5xFAD转基因小鼠出现相应的显著降低(p=0.01)。视网膜GSK3β和P-GSK3β含量变化与大脑表现一致。 结论 1.在发育和衰老过程中，5xFAD转基因小鼠和B6SJL非转基因小鼠视网膜和视神经上淀粉样前体蛋白、磷酸化淀粉样前体蛋白、β蛋白裂解酶、γ淀粉裂解酶、总tau蛋白和磷酸化tau蛋白PHF-1蛋白量的表达变化与大脑组织中的蛋白含量变化具有一致性。 2.淀粉样前体蛋白、磷酸化淀粉样前体蛋白、β蛋白裂解酶和γ淀粉裂解酶蛋白水平在9月龄5xFAD转基因小鼠视网膜和大脑组织中较同龄B6SJL非转基因小鼠和2月龄5xFAD转基因小鼠均有显著升高，而视紫红质蛋白水平则显著降低。 3.淀粉样前体蛋白、磷酸化淀粉样前体蛋白、β蛋白裂解酶、γ淀粉裂解酶和视紫红质在5xFAD转基因小鼠视网膜组织亚细胞结构中主要分布于内质网、高尔基体和早期内涵体中，与大脑组织亚细胞结构中的分布具有一致性。 4.总tau蛋白量、PHF-1蛋白量、PHF-1与总tau蛋白比值在9月龄5xFAD转基因小鼠视网膜和大脑组织中较同龄B6SJL非转基因小鼠和2月龄5xFAD小鼠均有显著升高； 5.5xFAD转基因小鼠视网膜上磷酸化tau蛋白的位点主要位于Ser396。 6. GSK3β信号通路有可能参与导致磷酸化tau蛋白PHF-1在老龄5xFAD转基因小鼠视网膜的蓄积。
[Abstract]:objective
1. of the amyloid precursor protein in 5xFAD transgenic mice eye with different structure protein distribution. Comparison of the amyloid precursor protein, phosphorylation of beta amyloid precursor protein, proteolytic enzymes, changes of protein gamma lyase in 5xFAD transgenic mice of different age groups and non transgenic mice B6SJL protein content in different structures of the eye ball. Observation of rhodopsin in the age, effect of different factors such as gene in 5xFAD transgenic mice and non transgenic mice B6SJL changes of retina.
2., we explored the distribution of amyloid precursor protein, phosphorylated amyloid precursor protein, beta amylase, gamma amylase and rhodopsin in the retinal tissue of 5xFAD transgenic mice.
3. of total tau protein and phosphorylated tau protein in 5xFAD transgenic mice eye with different structure protein distribution. To detect the expression of different sites of phosphorylation of tau protein in 5xFAD transgenic mice and non transgenic mice. B6SJL eye comparison of total tau protein and phosphorylated tau protein in 5xFAD transgenic mice of different age groups and B6SJL expression in transgenic mouse eye the change of protein.
4. detect the expression of GSK3 beta and P-GSK3 beta in different age groups 5xFAD transgenic mice and B6SJL non transgenic mice, and explore whether the GSK3 beta pathway is involved in the process of phosphorylation of tau protein in 5xFAD transgenic mice.
Method
1. selected 2 month old 5xFAD transgenic mice with Western Blot observation of amyloid precursor protein in cornea, lens, retina, optic nerve and brain tissue expression. Then 5xFAD transgenic mice of different age groups (February and 9 month old) and B6SJL non transgenic mice (1 weeks, January, February and 9 month old), the observation of WesternBlot starch kind of precursor protein, phosphorylation of beta amyloid precursor protein, proteolytic enzymes, protein gamma lyase and rhodopsin in mouse retina, optic nerve and brain tissue changes in protein expression.
2. selected 9 month old 5xFAD transgenic mice, with subcellular layered structure technology for the detection of amyloid precursor protein, phosphorylation of beta amyloid precursor protein, proteolytic enzymes, protein gamma lyase and rhodopsin in different subcellular structures such as endoplasmic reticulum, Golgi body distribution and early connotation.
3. selected 2 month old 5xFAD transgenic mice, using Western Blot to observe the total tau protein and phosphorylated tau protein in cornea, lens, retina, optic nerve and brain tissue expression. Then 5xFAD transgenic mice of different age groups (February and 9 month old (B6SJL) and non transgenic mice for 1 weeks, January, February and 9 month old) protein purification, and reduced phosphorylation after treatment, Western Blot observed similarities and differences expression of different phosphorylation of tau protein in the retina and brain of mice, and the total tau protein and phosphorylated tau protein in mouse retina, optic nerve and brain tissue protein expression changes.
4., 5xFAD transgenic mice and B6SJL non transgenic mice in different age groups (February and 9 month old) were selected. Western Blot was used to observe the relationship between the expression of GSK3 beta, P-GSK3 beta and phosphorylated tau protein in retina and brain tissues.
Result
1. amyloid precursor protein in 5xFAD transgenic mouse cornea, lens, retina, optic nerve were distributed, but different forms of expression, which is expressed in the retina and optic nerve on the protein form closest to the brain. The development period (1 weeks to 1 month old), amyloid precursor protein content in B6SJL gradually increase in transgenic mouse retina, and protein form mature. Adult (2 month old to 9 month old), amyloid precursor protein content in B6SJL transgenic mice and 5xFAD transgenic mice retina were significantly increased (p=0.02, P0.01).2 month old 5xFAD transgenic mice, retinal amyloid precursor protein and non transgenic B6SJL the mice had no significant difference, but the 9 month old, 5xFAD transgenic mouse retinal amyloid precursor protein content was significantly higher than that of non transgenic B6SJL mice (P0.01). The amyloid precursor protein content in non transgenic B6SJL In mice and 2 month old 5xFAD transgenic mice, the expression of amyloid precursor protein in the 9 month old 5xFAD transgenic mice was significantly higher than that in the transgenic mice (P0.01).
2. development period (1 weeks to 1 month old), the phosphorylation of the amyloid precursor protein content in non transgenic B6SJL mouse retina is low. In adulthood (2 month old to 9 month old), the phosphorylation of the amyloid precursor protein content in B6SJL non transgenic mice and 5xFAD transgenic mice retina were significantly increased (p=0.03, P0.01) and 2 month old and 9 month old, 5xFAD transgenic mouse retinal amyloid precursor protein phosphorylation levels were significantly higher than those of non transgenic B6SJL mice (P0.01, P0.01). The phosphorylation of the amyloid precursor protein content in B6SJL transgenic mice and 5xFAD transgenic mice 2 month old optic nerve was low, increased significantly at 9 month old 5xFAD transgenic mice (P0.01). The changes of body protein content of retinal samples of phosphorylated starch and brain.
3. development period (1 weeks to 1 month old), rhodopsin content increased significantly in B6SJL non transgenic mice retina. Adult (2 month old to 9 month old), the rhodopsin levels in B6SJL transgenic mice retina decreased, but decreased significantly in 5xFAD transgenic mouse retina (p=0.01).2 months, 5xFAD transgenic mice the retinal rhodopsin content and B6SJL non transgenic mice had no significant difference, but the 9 month old, 5xFAD transgenic mouse retina rhodopsin was significantly lower than that of non transgenic B6SJL mice (p=0.01).
4. development period (1 weeks to 1 month old), beta lyase and gamma lyase B6SJL starch content in non transgenic mice retina was significantly decreased. The adult stage (2 month old to 9 month old), beta lyase and gamma lyase starch content in B6SJL non retina transgenic mice and 5xFAD transgenic mice were significantly increased (P0.01, p=0.01). And the 2 month old and 9 month old, 5xFAD transgenic mouse retina beta lyase and gamma lyase starch content were significantly higher than non transgenic B6SJL mice (P0.01, P0.01). With the change of retina brain beta protein lyase and starch content of gamma lyase were consistent.
5.9 month old 5xFAD transgenic mice retina tissue subcellular localization showed amyloid precursor protein located in the endoplasmic reticulum, Golgi body and early endosomes, phosphorylation of amyloid beta protein in Golgi body, lytic enzymes located in the endoplasmic reticulum and early endosomes, gamma lyase protein located in the endoplasmic reticulum and Golgi, and rhodopsin in the ER net.
6. of the total tau protein and phosphorylated tau protein only distributed in 5xFAD transgenic mouse retina and optic nerve, consistent with the brain. In 5xFAD transgenic mice and non transgenic B6SJL mouse retina showed tau protein Ser396 phosphorylation site (PHF-1), and the brain.Ser202 was only consistent performance in 9 month old 5xFAD transgenic mouse retina and uncertain, and Ser199/Thr205 phosphorylation sites in the retina is not detected in the positive performance.
7.鍙戣偛鏈

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