環境壓力對人類神經細胞過度表現Protein Phosphatase 2A次單位PPP2R2B的影響

The Effect of Environmental Pressure on Human Neuronal Cells Overexpressing Protein Phosphatase 2A Subunit-PPP2R2B

鄭琬婷(Cheng Wan-Ting)

指導教授 ： 童麗珠 ； 方剛

國立臺灣師範大學/理學院/生命科學研究所/碩士(2009年)

摘要

Protein phosphatase 2A (PP2A)是屬於serine/threonine去磷酸酶的一種，能調控細胞的生長、存活和凋亡。哺乳類動物中，Bβ次單位是PP2A在腦神經細胞中的專一表現的次表型。當Bβ基因有所損害或過度表現時，會造成神經細胞的病變。本論文分別將能專一表現於細胞質的Bβ1和專一表現於粒線體的Bβ2質體轉殖入神經腫瘤母細胞SK-N-SH中，建立穩定細胞株。利用H2O2和tert-butyl hydroperoxide (tBHP)作為氧化壓力來源，經48小時的處理後，Bβ2細胞株的存活率會急遽下降，由Annexin V和PI雙重染色的結果中證實是因細胞凋亡而降低存活率，但Bβ1細胞株則否。進一步轉殖LC3-EGFP質體和對酸性液泡胞器染色鑑定細胞自噬的發生，可以看出Bβ2細胞株本身便有些微細胞自噬體存在，當經氧化壓力後更引發Bβ2細胞株有更多細胞自噬體出現。但以細胞自噬抑制劑3-methyladenine (3-MA)對細胞作前處理24小時後，再經過氧化物處理，則各Bβ2細胞株的sub-G1期細胞比例皆會下降，而酸性液泡胞器的密度也明顯降低。證明氧化壓力所引起的細胞凋亡是經由細胞自噬路徑而產生。另利用降低培養液中的血清濃度模擬營養缺乏的情況，當無血清培養48小時的Bβ1細胞株之sub-G1期細胞比例較Bβ2細胞株高出許多，當血清濃度降低時，Bβ1細胞株的酸性液泡胞器也較Bβ2細胞株的密度高，證實營養缺乏在Bβ1細胞株所引發的細胞凋亡是經由細胞自噬。本論文針對過度表現PPP2R2B的細胞株分別於氧化壓力和營養缺乏的壓力下，細胞所產生的生理變化，模擬神經退化病變的產生，並瞭解病變機制，祈能對神經退化疾病的研究與治療有所助益。

關鍵字

環境壓力 ； 神經細胞 ； PP2A ； 氧化壓力 ； 細胞自噬 ； 細胞凋亡

並列摘要

Protein phosphatase 2A (PP2A) is one of four major classes of serine/ threoine phosphatases. PP2A regulates in many cell functions, including growth, survival, and apoptosis. In mammals, the B family Bβ encodes a neuron-specific member of PP2A subunit with distinct expression patterns in brain. The defective and overexpressed Bβ can be associated with initiation of neurodegeneration. In this work, the construct of B subunit encoding cytoplasm-specific Bβ1 and mitochondria-specific Bβ2 was transfected into neuroblastoma cells, SK-N-SH, respectively, and the stable clones over-expressing either Bβ1 or Bβ2 were established. The cells were stimulated with oxidative pressure, H2O2 and tert-butyl hydroperoxide (t-BHP). Both H2O2 and tBHP reduced cell viabilities the Bβ2 clones after 48h, but not in Bβ1 clone. By Annexin V and propidium iodide staining, we have found that the apoptotic cell death in Bβ2 clones were increased in H2O2 and in tBHP depending on the clones used; whereas Bβ1 clones and the parental cells were unaffected. Using transient cells expressing autophagic marker, green fluorescent protein-tagged microtubule-associated protein light chain 3 (EGFP–LC3) and staining acidic vesicular organelles, we found that the induction of autophagy in Bβ2 clones is associated with apoptotic cell death under oxidative stress. The autophagy inhibitor, 3-methyladenine (3-MA), inhibits apoptosis and decreased acidic vesicular organelles induced by oxidative stress. On the other hand, compared to SK-N-SH cells, Bβ1 clones are more sensitive to serum deprivation by increasing sub-G1 population, while Bβ2 clones were less sensitive. This study offers more in-death knowledge on physiological changes of neuron cells overexpressing PPP2R2B under oxidative stress and nutrition deprivation.

並列關鍵字

Environmental Pressure ； Neuronal Cells ； Protein Phosphatase 2A ； Oxidative Stress ； Autophage ； Apoptosis

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