探討EGCG對第三型小腦脊髓運動失調症神經細胞株的保護作用

第三型小腦脊髓運動失調症為體染色體顯性遺傳的罕見疾病，屬於漸進性神經退化性疾病。此遺傳疾病主要是位於第14對染色體上ATAXIN-3基因的CAG核苷酸異常大量擴增，進而轉譯出具有大量麩氨酸的ataxin-3蛋白堆積，對細胞產生氧化壓力導致神經細胞的死亡。兒茶素(Epigallocatechin gallate, EGCG)是綠茶中含量最豐富的多酚類化合物具有抗氧化的功能，研究顯示對於細胞內高氧化壓力具有保護作用。在本篇論文中，首先利用神經腫瘤母細胞株SK-N-SH、以及先前已經構築能夠穩定表達全長突變ataxin-3蛋白的神經腫瘤母細胞，(SK-N-SH-MJD26、SK-N-SH-MJD78)，其三株細胞在EGCG 的前處理作用下，再利用過氧化氫(H2O2)的氧化作用觀察細胞對EGCG的抗氧化能力。MTT的實驗結果顯示在EGCG低劑量(2.5μM)的作用其細胞存活率明顯上升，在EGCG的前處理後再加入H2O2作用的細胞比起單獨加入過氧化氫作用時，其細胞的存活率卻是相對較提高。另一方面本實驗想了解EGCG的作用是如何保護細胞，於是以不同濃度EGCG (0, 1, 2.5, 5μM)作用於細胞後，利用西方點墨法觀察其熱休克蛋白(Heat shock protein)、細胞凋亡(apoptosis)以及細胞自噬 (autophagy)相關蛋白的表現情形。實驗結果顯示，三株細胞熱休克蛋白在EGCG的作用下，表現量均上升。在細胞凋亡蛋白方面，三株細胞抗凋亡蛋白Bcl2的表現量隨著EGCG濃度的增加而上升，而促細胞凋亡Bax的表現則有下降。此外，在細胞自噬作用的標記蛋白LC3II在EGCG的作用下，在三株細胞均大量表現。由以上實驗結果顯示EGCG能透過讓細胞內熱休克蛋白表現量的增加，減少細胞受到突變蛋白不正常堆積產生的壓力刺激。並且Bcl2表現量增加Bax表現量減少，顯示EGCG對細胞具有抗凋亡的傾向，且三株細胞LC3II大量的表現表示細胞亦產生自噬作用，啟動保護機制可能減緩神經細胞退化。

Spinocerebellar ataxia type 3 (SCA3), an autosomal dominant neurodegenerative disease, is caused by an increased number of CAG trinucleotide repeats in coding region of disease-associated gene ATAXIN-3 and thus results in a defective protein with a long polyQ track. In this study, neuroblastoma cell (SK-N-SH), was transduced to express either non-pathogenic ataxin-3-26Q or pathogenic ataxin-3-78Q protein. Three cell lines were treated with epigallocatechin gallate (EGCG) for 24 hr, H2O2 2hr and pre-treated with EGCG 24hr and then treated with H2O2 for 2hr. Cell viability were evaluated by the MTT assay. The results demonstrated that the cell viability were increased after treated with EGCG for 24 hr in these three cell lines, and the effect was dose dependent. In contrast, the cell viability was decreased after treated with H2O2 for 2hr. The viability were realatively increased after pre-treated with EGCG for 24hr and then treated with H2O2 for 2hr. On the other hand, to investigate how EGCG protect the neuron cells, heat shock protein, apoptosis relative proteins and autophagy relative proteins were analyzed by Western blot. Our data indicated that Hsp were up-regulated after treated with EGCG in three cell lines. Anti-apoptotic protein, Bcl-2 was up- regulated and pro-apoptotic protein, Bax was down-regulated, autophagy marker protein, LC3 were up-regulated. In conclusion, our results displayed that EGCG might protect the cell through up-regulate the level of Hsp to help cleaning up the misfolding aggregated proteins in the cells, possessing the effect of anti-apoptosis, and up regulating autophagy marker protein.