發(fā)布時(shí)間：2018-03-02 08:42

  本文關(guān)鍵詞： 衰老 淀粉樣前體蛋白 tau蛋白 磷酸化 5xFAD小鼠　出處：《中國(guó)人民解放軍醫(yī)學(xué)院》2013年博士論文　論文類型：學(xué)位論文

【摘要】：目的 1.研究淀粉樣前體蛋白在5xFAD轉(zhuǎn)基因小鼠眼球不同結(jié)構(gòu)的蛋白分布。比較淀粉樣前體蛋白、磷酸化淀粉樣前體蛋白、β蛋白裂解酶、γ蛋白裂解酶在不同年齡組5xFAD轉(zhuǎn)基因小鼠和B6SJL非轉(zhuǎn)基因小鼠眼球不同結(jié)構(gòu)中蛋白含量的變化。觀察視紫紅質(zhì)在年齡、基因等不同因素作用下在5xFAD轉(zhuǎn)基因小鼠和B6SJL非轉(zhuǎn)基因小鼠視網(wǎng)膜的變化。 2．探索淀粉樣前體蛋白、磷酸化淀粉樣前體蛋白、β淀粉裂解酶、γ淀粉裂解酶和視紫紅質(zhì)在5xFAD轉(zhuǎn)基因小鼠視網(wǎng)膜組織細(xì)胞亞結(jié)構(gòu)的分布。 3.研究總tau蛋白和磷酸化tau蛋白在5xFAD轉(zhuǎn)基因小鼠眼球不同結(jié)構(gòu)的蛋白分布。檢測(cè)不同位點(diǎn)磷酸化tau蛋白在5xFAD轉(zhuǎn)基因小鼠和B6SJL非轉(zhuǎn)基因小鼠眼球的表達(dá)。比較總tau蛋白和磷酸化tau蛋白在不同年齡組5xFAD轉(zhuǎn)基因小鼠和B6SJL非轉(zhuǎn)基因小鼠眼球蛋白表達(dá)的改變。 4.檢測(cè)GSK3β和P-GSK3β含量在不同年齡組5xFAD轉(zhuǎn)基因小鼠和B6SJL非轉(zhuǎn)基因小鼠眼球蛋白表達(dá)的改變，探討GSK3β通路是否參與磷酸化tau蛋白在5xFAD轉(zhuǎn)基因小鼠眼部病變的過(guò)程。 方法 1.選取2月齡5xFAD轉(zhuǎn)基因小鼠，用Western Blot觀察淀粉樣前體蛋白在角膜、晶狀體、視網(wǎng)膜、視神經(jīng)和大腦組織中的表達(dá)。再選取不同年齡組5xFAD轉(zhuǎn)基因小鼠（2月和9月齡）和B6SJL非轉(zhuǎn)基因小鼠（1周、1月、2月和9月齡），WesternBlot觀察淀粉樣前體蛋白、磷酸化淀粉樣前體蛋白、β蛋白裂解酶、γ蛋白裂解酶和視紫紅質(zhì)在小鼠視網(wǎng)膜、視神經(jīng)和大腦組織蛋白表達(dá)的改變。 2.選取9月齡5xFAD轉(zhuǎn)基因小鼠，用組織亞細(xì)胞結(jié)構(gòu)分層技術(shù)探測(cè)淀粉樣前體蛋白、磷酸化淀粉樣前體蛋白、β蛋白裂解酶、γ蛋白裂解酶和視紫紅質(zhì)在不同亞細(xì)胞結(jié)構(gòu)如內(nèi)質(zhì)網(wǎng)、高爾基體和早期內(nèi)涵體的分布。 3.選取2月齡5xFAD轉(zhuǎn)基因小鼠，用Western Blot觀察總tau蛋白和磷酸化tau蛋白在角膜、晶狀體、視網(wǎng)膜、視神經(jīng)和大腦組織中的表達(dá)。再選取不同年齡組5xFAD轉(zhuǎn)基因小鼠（2月和9月齡）和（B6SJL非轉(zhuǎn)基因小鼠1周、1月、2月和9月齡），蛋白純化并降磷酸化處理后，Western Blot觀察不同位點(diǎn)磷酸化tau蛋白在小鼠視網(wǎng)膜和大腦表達(dá)的異同，以及總tau蛋白和磷酸化tau蛋白在小鼠視網(wǎng)膜、視神經(jīng)和大腦組織上蛋白表達(dá)的改變。 4.選取不同年齡組（2月和9月齡）5xFAD轉(zhuǎn)基因小鼠和B6SJL非轉(zhuǎn)基因小鼠，Western Blot觀察GSK3β，P-GSK3β與磷酸化tau蛋白在視網(wǎng)膜和大腦組織上蛋白表達(dá)變化的關(guān)系。 結(jié)果 1.淀粉樣前體蛋白在5xFAD轉(zhuǎn)基因小鼠的角膜、晶狀體、視網(wǎng)膜、視神經(jīng)上均有分布，但表達(dá)形式不同，其中表達(dá)于視網(wǎng)膜和視神經(jīng)上的蛋白形態(tài)最接近于大腦。發(fā)育期(1周齡至1月齡)，淀粉樣前體蛋白含量在B6SJL非轉(zhuǎn)基因小鼠視網(wǎng)膜上逐漸增加，且蛋白形式趨于成熟。成年期(2月齡至9月齡)，淀粉樣前體蛋白含量在B6SJL非轉(zhuǎn)基因小鼠和5xFAD轉(zhuǎn)基因小鼠視網(wǎng)膜上均顯著升高(p=0.02, p0.01)。2月齡時(shí)，5xFAD轉(zhuǎn)基因小鼠視網(wǎng)膜淀粉樣前體蛋白含量與B6SJL非轉(zhuǎn)基因小鼠無(wú)明顯差異，但9月齡時(shí)，5xFAD轉(zhuǎn)基因小鼠視網(wǎng)膜淀粉樣前體蛋白含量顯著高于B6SJL非轉(zhuǎn)基因小鼠(p0.01)。淀粉樣前體蛋白含量在B6SJL非轉(zhuǎn)基因小鼠和2月齡5xFAD轉(zhuǎn)基因小鼠視神經(jīng)上均較低，在9月齡5xFAD轉(zhuǎn)基因小鼠顯著升高(p0.01)。視網(wǎng)膜淀粉樣前體蛋白含量變化與大腦表現(xiàn)一致。 2.發(fā)育期(1周齡至1月齡)，磷酸化淀粉樣前體蛋白含量在B6SJL非轉(zhuǎn)基因小鼠視網(wǎng)膜上較低。成年期(2月齡至9月齡)，磷酸化淀粉樣前體蛋白含量在B6SJL非轉(zhuǎn)基因小鼠和5xFAD轉(zhuǎn)基因小鼠視網(wǎng)膜上均顯著升高(p=0.03, p0.01)。且2月齡和9月齡時(shí)，5xFAD轉(zhuǎn)基因小鼠視網(wǎng)膜磷酸化淀粉樣前體蛋白含量均顯著高于B6SJL非轉(zhuǎn)基因小鼠(p0.01, p0.01)。磷酸化淀粉樣前體蛋白含量在B6SJL非轉(zhuǎn)基因小鼠和2月齡5xFAD轉(zhuǎn)基因小鼠視神經(jīng)上均較低，在9月齡5xFAD轉(zhuǎn)基因小鼠顯著升高(p0.01)。視網(wǎng)膜磷酸化淀粉樣前體蛋白含量變化與大腦表現(xiàn)一致。 3.發(fā)育期(1周齡至1月齡)，視紫紅質(zhì)含量在B6SJL非轉(zhuǎn)基因小鼠視網(wǎng)膜顯著增加。成年期(2月齡至9月齡)，視紫紅質(zhì)含量在B6SJL非轉(zhuǎn)基因小鼠視網(wǎng)膜有所下降，但在5xFAD轉(zhuǎn)基因小鼠視網(wǎng)膜下降顯著(p=0.01）。2月齡時(shí)，5xFAD轉(zhuǎn)基因小鼠視網(wǎng)膜視紫紅質(zhì)含量與B6SJL非轉(zhuǎn)基因小鼠無(wú)明顯差異，但9月齡時(shí)，5xFAD轉(zhuǎn)基因小鼠視網(wǎng)膜視紫紅質(zhì)含量顯著低于B6SJL非轉(zhuǎn)基因小鼠(p=0.01）。 4.發(fā)育期(1周齡至1月齡)，β蛋白裂解酶和γ淀粉裂解酶含量在B6SJL非轉(zhuǎn)基因小鼠視網(wǎng)膜上顯著降低。成年期(2月齡至9月齡)，β蛋白裂解酶和γ淀粉裂解酶含量在B6SJL非轉(zhuǎn)基因小鼠和5xFAD轉(zhuǎn)基因小鼠視網(wǎng)膜上均顯著升高（p0.01,p=0.01）。且2月齡和9月齡時(shí)，5xFAD轉(zhuǎn)基因小鼠視網(wǎng)膜β蛋白裂解酶和γ淀粉裂解酶含量均顯著高于B6SJL非轉(zhuǎn)基因小鼠（p0.01, p0.01）。視網(wǎng)膜β蛋白裂解酶和γ淀粉裂解酶含量變化與大腦表現(xiàn)一致。 5.9月齡5xFAD轉(zhuǎn)基因小鼠視網(wǎng)膜組織亞細(xì)胞結(jié)構(gòu)定位顯示淀粉樣前體蛋白位于內(nèi)質(zhì)網(wǎng)、高爾基體和早期內(nèi)涵體，磷酸化淀粉樣蛋白位于高爾基體，β蛋白裂解酶位于內(nèi)質(zhì)網(wǎng)和早期內(nèi)涵體，，γ蛋白裂解酶位于內(nèi)質(zhì)網(wǎng)和高爾基體，而視紫紅質(zhì)位于內(nèi)質(zhì)網(wǎng)。 6.總tau蛋白和磷酸化tau蛋白僅在5xFAD轉(zhuǎn)基因小鼠的視網(wǎng)膜和視神經(jīng)上有分布，與大腦表現(xiàn)一致。在5xFAD轉(zhuǎn)基因小鼠和B6SJL非轉(zhuǎn)基因小鼠視網(wǎng)膜上主要表現(xiàn)為tau蛋白Ser396磷酸化位點(diǎn)(PHF-1)，與大腦表現(xiàn)一致。Ser202位點(diǎn)僅表現(xiàn)在9月齡5xFAD轉(zhuǎn)基因小鼠視網(wǎng)膜且不確定，而Ser199/Thr205磷酸化位點(diǎn)在視網(wǎng)膜上則未檢測(cè)到陽(yáng)性表現(xiàn)。 7.發(fā)育期(1周齡至1月齡)，總tau蛋白含量在B6SJL非轉(zhuǎn)基因小鼠視網(wǎng)膜上顯著增加。成年期(2月齡至9月齡)，總tau蛋白含量在B6SJL非轉(zhuǎn)基因小鼠和5xFAD轉(zhuǎn)基因小鼠視網(wǎng)膜上均顯著升高(p0.01, p0.01)。2月齡和9月齡時(shí)，5xFAD轉(zhuǎn)基因小鼠視網(wǎng)膜總tau蛋白含量均顯著高于B6SJL非轉(zhuǎn)基因小鼠(p0.01, p0.01)。總tau蛋白含量在B6SJL非轉(zhuǎn)基因小鼠和2月齡5xFAD轉(zhuǎn)基因小鼠視神經(jīng)上均較低，在9月齡5xFAD轉(zhuǎn)基因小鼠顯著升高。視網(wǎng)膜總tau蛋白含量變化與大腦表現(xiàn)一致。 8.發(fā)育期(1周齡至1月齡)，PHF-1含量和PHF-1與總tau蛋白含量的比值在B6SJL非轉(zhuǎn)基因小鼠視網(wǎng)膜上顯著較低。成年期(2月齡至9月齡)，PHF-1含量和PHF-1與總tau蛋白含量的比值在B6SJL非轉(zhuǎn)基因小鼠視網(wǎng)膜上呈增高趨勢(shì)，在轉(zhuǎn)基因5xFAD小鼠視網(wǎng)膜上呈顯著增高(p0.01, p0.01)。且2月齡時(shí)，5xFAD轉(zhuǎn)基因小鼠視網(wǎng)膜PHF-1含量和PHF-1與總tau蛋白含量的比值與B6SJL非轉(zhuǎn)基因小鼠無(wú)差異，但9月齡時(shí)，5xFAD轉(zhuǎn)基因小鼠視網(wǎng)膜PHF-1含量和PHF-1與總tau蛋白含量的比值均顯著高于B6SJL非轉(zhuǎn)基因小鼠(p0.01, p0.01)。視網(wǎng)膜PHF-1含量變化與大腦表現(xiàn)一致。 9.成年期(2月齡至9月齡)，GSK3β含量在9月齡5xFAD轉(zhuǎn)基因小鼠視網(wǎng)膜較同齡B6SJL非轉(zhuǎn)基因小鼠和2月齡5xFAD轉(zhuǎn)基因小鼠顯著升高(p=0.02)，而P-GSK3β含量在9月齡5xFAD轉(zhuǎn)基因小鼠視網(wǎng)膜較同齡B6SJL非轉(zhuǎn)基因小鼠和2月齡5xFAD轉(zhuǎn)基因小鼠出現(xiàn)相應(yīng)的顯著降低(p=0.01)。視網(wǎng)膜GSK3β和P-GSK3β含量變化與大腦表現(xiàn)一致。 結(jié)論 1.在發(fā)育和衰老過(guò)程中，5xFAD轉(zhuǎn)基因小鼠和B6SJL非轉(zhuǎn)基因小鼠視網(wǎng)膜和視神經(jīng)上淀粉樣前體蛋白、磷酸化淀粉樣前體蛋白、β蛋白裂解酶、γ淀粉裂解酶、總tau蛋白和磷酸化tau蛋白PHF-1蛋白量的表達(dá)變化與大腦組織中的蛋白含量變化具有一致性。 2.淀粉樣前體蛋白、磷酸化淀粉樣前體蛋白、β蛋白裂解酶和γ淀粉裂解酶蛋白水平在9月齡5xFAD轉(zhuǎn)基因小鼠視網(wǎng)膜和大腦組織中較同齡B6SJL非轉(zhuǎn)基因小鼠和2月齡5xFAD轉(zhuǎn)基因小鼠均有顯著升高，而視紫紅質(zhì)蛋白水平則顯著降低。 3.淀粉樣前體蛋白、磷酸化淀粉樣前體蛋白、β蛋白裂解酶、γ淀粉裂解酶和視紫紅質(zhì)在5xFAD轉(zhuǎn)基因小鼠視網(wǎng)膜組織亞細(xì)胞結(jié)構(gòu)中主要分布于內(nèi)質(zhì)網(wǎng)、高爾基體和早期內(nèi)涵體中，與大腦組織亞細(xì)胞結(jié)構(gòu)中的分布具有一致性。 4.總tau蛋白量、PHF-1蛋白量、PHF-1與總tau蛋白比值在9月齡5xFAD轉(zhuǎn)基因小鼠視網(wǎng)膜和大腦組織中較同齡B6SJL非轉(zhuǎn)基因小鼠和2月齡5xFAD小鼠均有顯著升高； 5.5xFAD轉(zhuǎn)基因小鼠視網(wǎng)膜上磷酸化tau蛋白的位點(diǎn)主要位于Ser396。 6. GSK3β信號(hào)通路有可能參與導(dǎo)致磷酸化tau蛋白PHF-1在老齡5xFAD轉(zhuǎn)基因小鼠視網(wǎng)膜的蓄積。
[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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