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研究生:林巧梅
研究生(外文):Chiao-Mei Lin
論文名稱:海馬迴細胞培養中ProteinIneractingwithC-kinase(PICK1)之分佈
論文名稱(外文):Intracellular Distribution of Protein Interacitng with C-kinase in Cultured Hippocampal Cells
指導教授:周韻家
指導教授(外文):Yun-Chia Chou
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:神經科學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
中文關鍵詞:海馬迴神經細胞培養海馬迴星狀神經膠細胞培養麩胺酸細胞內分佈
外文關鍵詞:hippocampal neuron culturehippocampal astrocyte cultureglutamatePICK1intracellular distribution
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Protein interacting with C-kinase (PICK1)是一種PDZ-containing protein,最初是利用protein kinase C (PKC)α進行酵母菌雙雜合系統而被發現到。目前已知,PICK1可以與麩胺酸受器AMPA(α-amino-3-hydroxyl -5-methyl-4- isoxazole propionic acid)receptor結合,在幫助AMPA receptor在突觸的細胞膜之定位上扮演一個重要的角色。另外PICK1還可幫助PKCα與AMPA receptor結合,以便AMPA receptor被磷酸化。之前已有研究指出,麩胺酸之興奮性毒性會透過AMPA receptor以及活化PKC,因此我們主要在探討麩胺酸長時間的刺激之下,是否會影響體外培養的海馬迴神經細胞與星狀神經膠細胞內的PICK1之表現及其細胞內分佈。
我們發現利用麩胺酸長時間處理體外培養的海馬迴神經細胞後,減少了細胞質PICK1之表現量,但不會影響細胞膜PICK1之表現量。就整體PICK1表現量而言,麩胺酸對整體PICK1之表現量也沒有顯著地影響,因此我們推測也許麩胺酸造成PICK1轉位積聚在細胞核中。經由細胞免疫染色結果顯示在麩胺酸處理後海馬迴神經細胞之細胞核中會有PICK1之大量積聚。另外也發現隨著麩胺酸處理的時間越久,細胞核中具有PICK1積聚之細胞數目越多,而且乳酸脫氫酶之釋放程度也越嚴重。這些細胞核中有PICK1積聚之細胞,均無法為microtubule-associated protein 2(MAP2)以及 glial fibrillary acidic protein(GFAP)之抗體所標示。另一方面,我們發現在麩胺酸處理6小時之後再經回復期8小時,同樣造成細胞質PICK1之表現量減少,但不影響細胞膜以及整體PICK1之表現量。利用細胞免疫染色更進一步証實在回復期後,細胞核中仍然有PICK1之積聚,此意謂著麩胺酸處理之後所觀察到之PICK1在細胞核中積聚的現象並非經由一非特異性的機制。這些結果顯示PICK1可能在細胞死亡中扮演一個特殊的調控角色。
另外,我們發現將海馬迴神經細胞培養在缺少生長因子的情形下,也會造成PICK1在細胞質的表現量減少,但是這個現象沒有麩胺酸處理所看到的情況顯著。然而對海馬迴星狀神經膠細胞而言,麩胺酸處理並不會影響PICK1的表現及其細胞內分佈。總而言之,PICK1在海馬迴神經細胞細胞死亡的機制中扮演一個重要的角色,而且PICK1在神經細胞及星狀神經膠細胞這二種細胞中所扮演的角色並不相同。
Protein interacting with C-kinase (PICK1), a PDZ-containing protein, is initially identified as a protein kinase C (PKC) α-binding protein by the yeast two-hybrid screening. It has been known to interact with the AMPA (α-amino- 3-hydroxy-5-methyl-4-isoxazole propionic acid) subtype of glutamate receptor. Previous work suggests that PICK1 plays a role in the synaptic localization of AMPA receptor and it can target PKCα to phosphorylate AMPA receptor. Since glutamate-induced excitotoxicity is mediated through glutamate receptors and PKCα, we therefore examined whether glutamate affected the expression and intracellular distribution of PICK1 in cultured hippocampal neurons and astrocytes.
Glutamate decreased the level of cytosolic PICK1 while having no effect on that of membranous PICK1 in neuronal cultures. Since glutamate had no significant effect on the total amount of cellular PICK1, it was speculated that glutamate might result in accumulation of PICK1 in nuclei. Immunocytochemical staining study revealed nuclear accumulation of PICK1 in neuronal cultures after glutamate exposure. There was an increase in nuclear accumulation of PICK1 and the release of lactate dehydrogenase (LDH) with the exposure time of glutamate. These cells with nuclear accumulation of PICK1 were neither microtubule-associated protein 2 nor glial fibrillary acidic protein immunoreactive. After a 6-hr exposure to glutamate, we also found that the level of cytosolic PICK1 was still decreased and there was no change in the level of membranous and total amount of PICK1 during the subsequent 8-h ’recovery’ period. Immunocytochemical staining further confirmed nuclear accumulation of PICK1 during the ’recovery’ period, suggesting that glutamate-induced nuclear accumulation of PICK1 was not via a non-specific leakage mechanism. These results reveal that PICK1 plays a specifically regulatory role in cell death.
We also found decreased level of cytosolic PICK1 in neurons treated with buffer containing no growth factor, though at a degree less than that observed in neurons exposed to glutamate. However, glutamate had no effect on the expression and intracellular distribution of PICK1 in astrocyte cultures. In conclusion, this study provides evidence supporting the notion that PICK1 involved in the mechanism of cell death in hippocampal neuronal culture. Moreover, the intracellular distribution of PICK1 is regulated in a different manner in astrocytes.
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