本文選題：Spy1 + CRMP1��； 參考：《南通大學》2014年碩士論文

【摘要】：目的驗證Spy1與CRMP1的相互作用,分析Spy1對于CRMP1的磷酸化水平調節(jié)在Sema3A誘導的生長錐塌陷過程中的影響,探討兩者相互作用與神經元再生之間的關系。方法1.運用免疫共沉淀驗證Spy1和CRMP1(collapsin response mediator proteins)之間的相互作用關系;構建Spy1和CRMP1截短突變質粒,轉染至工具細胞中,檢測其相互作用結構域;同時運用免疫熒光雙標檢測Spy1和CRMP1在神經元中的共定位情況;免疫共沉淀檢測CRMP1與actin的關系;Spy1/CRMP1對于細胞骨架動態(tài)性影響。2.Western blot檢測Spy1及CRMP1在Sem3A誘導的生長錐塌陷中的表達變化;構建CRMP1點突變質粒,轉染原代DRG神經元,分析在Sema3A誘導的生長錐塌陷過程中對于神經元生長錐形態(tài)的影響。結果1.在PC12未分化細胞中Spy1和CRMP1存在相互作用;免疫熒光顯示Spy1和CRMP1在未分化的PC12中都存在于胞漿;NGF(100ng/ml)刺激PC12未分化細胞分化和原代培養(yǎng)DRG神經元過程中都觀察到Spy1和CRMP1在突起末端和邊緣的聚集。2.HEK293T工具細胞中發(fā)現(xiàn)Spy1域與CRMP1的530~572結構域結合;Spy1可有效的增強CDK5的激酶活性,從而介導對于CRMP1的磷酸化調節(jié)作用。3.發(fā)現(xiàn)CRMP1與actin存在相互作用;Spy1對CRMP1的磷酸化的增強效應會干擾CRMP1與actin的相互作用,從而影響細胞骨架的動態(tài)性,并介導Sema3A的生長錐塌陷效應;體外培養(yǎng)的DRG神經元用Sema3A刺激后發(fā)現(xiàn)明顯的生長錐塌陷現(xiàn)象;體外siSpy1病毒感染原代DRG神經元并進行Sema3A刺激,發(fā)現(xiàn)生長錐的塌陷受到明顯的抑制;而siSpy1與CRMP1的擬磷酸化點突變質粒CRMP1T509D或CRMP1S522D共同感染并在Sema3A刺激下,發(fā)現(xiàn)生長錐的塌陷現(xiàn)象得到部分回復。結論1.Spy1與CRMP1存在相互作用,Spy1可通過增強CDK5的激酶活性從而增強對CRMP1的磷酸化調節(jié)。2.CRMP1與actin存在相互作用;而CRMP1的高磷酸化水平會干擾CRMP1與actin的相互作用,從而影響細胞骨架的動態(tài)性,介導Sema3A誘導的生長錐塌陷效應。3.CRMP1的磷酸化點T509和S522介導了Sema3A誘導的生長錐塌陷效應。
[Abstract]:Aim to verify the interaction between Spy1 and CRMP1, to analyze the effect of Spy1 on the phosphorylation of CRMP1 in the process of Sema3A induced growth cone collapse, and to explore the relationship between the interaction and neuronal regeneration. Method 1. The interaction relationship between Spy1 and CRMP1(collapsin response mediator proteins was verified by immunoprecipitation, and the truncated mutant plasmids of Spy1 and CRMP1 were constructed and transfected into tool cells to detect their interaction domains. At the same time, the co-localization of Spy1 and CRMP1 in neurons was detected by immunofluorescence double labeling, and the relationship between CRMP1 and actin was detected by immunoprecipitation. The effect of Spy1 / CRMP1 on cytoskeleton dynamics was detected. 2. Western blot was used to detect the expression of Spy1 and CRMP1 in Sem3A induced growth cone collapse. CRMP1 point mutation plasmid was constructed and transfected into primary DRG neurons. The effect of Sema3A induced growth cone collapse on the morphology of neuron growth cone was analyzed. Result 1. There was interaction between Spy1 and CRMP1 in PC12 undifferentiated cells. Immunofluorescence showed that both Spy1 and CRMP1 were present in undifferentiated PC12 (100 ng / ml) stimulated PC12 undifferentiated cell differentiation and primary culture of DRG neurons. Both Spy1 and CRMP1 were observed in the aggregation of end and edge of protuberance. 2. HEK293T tool cells. The binding of Spy1 domain and CRMP1 530t572 domain to Spy1 can effectively enhance the activity of CDK5 kinase. Thus mediates the phosphorylation regulation of CRMP1. It was found that there was interaction between CRMP1 and actin. The enhancement effect of Spy1 on the phosphorylation of CRMP1 would interfere with the interaction between CRMP1 and actin, thus affecting the dynamics of cytoskeleton and mediating the collapse of Sema3A growth cone. DRG neurons cultured in vitro showed obvious collapse of growth cone after stimulation with Sema3A, and siSpy1 virus infected primary DRG neurons and stimulated with Sema3A in vitro, and found that the collapse of growth cone was obviously inhibited. However, siSpy1 and CRMP1 were co-infected with CRMP1T509D or CRMP1S522D, and under the stimulation of Sema3A, the collapse of growth cone was partially recovered. Conclusion there is interaction between 1.Spy1 and CRMP1. Spy1 can enhance the phosphorylation regulation of CRMP1 by enhancing the activity of CDK5 kinase. 2. CRMP1 interacts with actin, while the high phosphorylation level of CRMP1 interferes with the interaction between CRMP1 and actin. Therefore, the cytoskeleton dynamics was affected, and the growth cone collapse induced by Sema3A was mediated. 3. The phosphorylation point T509 and S522 of CRMP1 mediated the collapse of growth cone induced by Sema3A.
【學位授予單位】：南通大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R745
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本文編號：1781922