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研究生:王之奎
研究生(外文):Chih-Kuei Wang
論文名稱:PP2A在視網膜節細胞凋亡中所扮演的角色
論文名稱(外文):The role of PP2A in the apoptosis of retinal ganglion cells
指導教授:王安國王安國引用關係
指導教授(外文):An-Guor Wang
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:2009
畢業學年度:97
語文別:中文
論文頁數:43
中文關鍵詞:視網膜節細胞第二型蛋白磷酸水解酶
外文關鍵詞:retinal ganglion cellprotein phosphatase 2A
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視網膜節細胞可以聚集感光細胞所接收的光訊號並傳遞至大腦,在視覺訊息的傳導中扮演很重要的角色。視神經節細胞如果因疾病或傷害而死亡,將導致嚴重的視力損害。因此,在眼科研究中研究視網膜節細胞的死亡機制一直是一個熱門課題。本篇文章中,我們利用視神經節細胞的一種細胞株RGC-5,來探討蛋白質去磷酸酶protein phosphatase 2A (PP2A)在視網膜節細胞缺血清凋亡中所扮演的角色。我們將RGC-5培養在無血清的培養基中,部份細胞會發生凋亡現象。將PP2A的活性以其抑制劑endothall抑制後,會促進由血清缺乏所造成的細胞凋亡,並導致caspase 3的切割,同時也造成磷酸化Akt和ERK的減少。我們想探討endothall是否是透過抑制Akt和ERK促進細胞凋亡,因此我們分別加入抑制Akt和ERK磷酸化的抑制劑LY294002和PD98059。PD98059可以完全抑制ERK的磷酸化,但不會促進細胞死亡,顯示endothall應該不是透過抑制ERK促進細胞凋亡。LY294002可以完全抑制Akt的磷酸化,同時也會促進細胞死亡。然而我們以胰島素處理或是基因轉染技術恢復Akt的活性卻無法達到保護細胞的效果,顯示Akt可能不是唯一受到PP2A抑制的影響而造成細胞凋亡的蛋白質。我們也測試了p53參與其中的可能性,磷酸化的p53(Ser15)和所有的p53都在PP2A抑制後而增加,但是加入可以抑制p53轉錄功能的抑制劑pifithrin-脉對RGC-5細胞也無保護作用。這顯示p53可能也不是主要造成RGC-5死亡的蛋白質。關於PP2A抑制所促進RGC-5細胞缺血清凋亡的主要機轉仍需做進一步的研究及探討。
Retinal ganglion cell plays a critical role in vision because of its property of relaying signal from photoreceptors to visual cortex. Many researchers had been devoted to investigate how retinal ganglion cell die in diseases or stresses. In this study we used a transformed retinal ganglion cell, RGC-5, to study if protein phosphatase 2A (PP2A) participated in serum deprivation-induced apoptosis. We found that inhibition of PP2A by endothall promotes caspase 3 cleavage and apoptosis of RGC-5. It also decreases the amount of phosphorylated Akt and ERK. We had used two drugs to block the phosphorylation of ERK and Akt. PD98059, an inhibitor for the phosphorylation of ERK, does not promote apoptosis in serum-deprived RGC-5. It indicates that PP2A inhibition may not promote RGC-5 apoptosis by dephosphorylating ERK. LY294002, an inhibitor for the phosphorylation of Akt, promotes the serum-deprived apoptosis in RGC-5. However, rescuing Akt by insulin treatment or myr-Akt transfection does not protect RGC-5, suggesting that Akt maybe is not the only protein regulating apoptosis induced by PP2A inhibition. p53 is another protein possibly involved in PP2A inhibition-induced apoptosis. PP2A inhibition increases the amount of phspho-p53 (Ser15) and total p53. Pifithrin-脉, a transcriptional inhibitor of p53, is unable to protect RGC-5. This result suggests that p53 is not the major protein in PP2A inhibition-induced apoptosis. The mechanism in PP2A inhibition promoting serum-deprived RGC-5 apoptosis still needs further investigation.
Abstract (In Chinese)
Abstract
Table of contents
1. Introduction…………………………………………………………………..5
1.1. Retina and retinal ganglion cells (RGCs)
1.2. The disease affecting retinal ganglion cells.
1.3. Transformed retinal ganglion cell (RGC5)
1.4. Apoptosis
1.5. Protein phosphatase 2A (PP2A)
1.6. PP2A and apoptosis
2. Materials and Methods……………………………………………………..10
2.1. Reagent and antibodies
2.2. Cell culture
2.3. PP2A activity assay
2.4. Serum deprivation treatment
2.5. Cytotoxicity assay (LDH releasing assay)
2.6. DAPI staining
2.7. DNA ladder assay
2.8. Immunoblot assay
2.9. Gene construct and transfection
2.10. Statistics
3. Results……………………………………………………………………….15
3.1. Endothall inhibits the activity of PP2A in RGC-5
3.2. PP2A inhibition promotes serum deprivation-induced cell death in RGC-5
3.3. PP2A inhibition promotes RGC-5 cell death by apoptosis
3.4. PP2A inhibition promotes caspase 3 cleavage
3.5. PP2A inhibition decreases phosphorylated ERK and Akt
3.6. Inhibition of ERK phosphorylation does not promote the apoptosis of RGC-5
3.7. ERK can not be phosphorylated after transfection of constitutively active MEK in RGC-5
3.8. Inhibition of Akt pathway promotes RGC-5 cell death
3.9. Rescue of Akt has no protective effect on RGC-5
3.10. p53 is not the key factor of apoptosis caused by PP2A inhibition
4. Discussion…………………………………………………………………...20
4.1. Endothall inhibits the enzyme activity of PP2A and promotes cell death of RGC-5
4.2. PP2A inhibition results in cell apoptosis and caspase 3 cleavage in serum deprived RGC-5
4.3. PP2A inhibition decreases the amount of phosphorylated Akt and ERK
4.4. The role of p53 in PP2A inhibition-induced apoptosis
5. References…………………………………………………………………...24
6. Figures……………………………………………………………………….30
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