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標題: | SH-SY5Y中Pink1的神經保護作用之探討 Neuroprotective action of Pink1 in SH-SY5Y neuroblastoma cells |
作者: | Tzeng-Ruei Lee 李政叡 |
指導教授: | 符文美(Wen-Mei Fu) |
關鍵字: | 巴金森氏症, Pink1,HO-1, |
出版年 : | 2008 |
學位: | 碩士 |
摘要: | 巴金森氏症是當今最常見的神經退化性疾病之一,以目前的藥物治療上只能緩解症狀而不能根除。目前對於其神經退化的致病機轉仍不清楚。Pink1是早發性巴金森氏症的隱性基因,Pink1的突變與否對早發性巴金森氏症來說非常重要。我們在SH-SY5Y中,做了Pink1變異的三個G309D clones和二個P209A clones共五個mutant stable clones,並和wild-type比較,發現用H2O2所引發的細胞內ROS的增加或細胞的凋亡,Pink1 mutant stable clones都有明顯的增加現象。而在dopamine uptake方面,給予H2O2後,Pink1 mutant stable clones的dopamine uptake減少的比wild-type更為明顯。另外利用BrdU Assay發現Pink1 mutant stable clones的增生速率要比wild-type慢很多。
Heme oxygenase 1 (HO-1) 是細胞遇到氧化壓力時產生的一個細胞保護分子,我們因此觀察了細胞經由H2O2所引發的HO-1的變化。在mRNA的部份,wild-type在一般情況的含量較低,但卻很容易被H2O2所引發 ; 而Pink1 mutant stable clones中的一些clones雖然在靜止狀態中的含量較高,但是在給予H2O2後HO-1 mRNA卻完全無法被引發。而在HO-1蛋白的部分則有類似的情況。接著我們用virus infection的方法使SH-SY5Y大量表現HO-1蛋白,發現Pink1 mutant stable clones經由H2O2所造成的細胞凋亡加速現象可以被拮抗。根據以上的結果,我們推測Pink1在細胞內的作用機制可能和HO-1有關,Pink1有可能參與了HO-1的引發路徑。此外,我們也觀察了細胞經由H2O2所引發的BDNF mRNA的變化。我們發現到大部份的Pink1 mutant stable clones在basal condition下的BDNF mRNA level都比wild-type高 ; 而給予H2O2後則在wild-type或是Pink1 mutant stable clones中都可以看到BDNF mRNA level下降的現象。我們初步推測BDNF似乎不是影響Pink1 mutant stable clones在遭受刺激後提升apoptotic rate的因子。 我們的結果發現Pink1在G309D或P209A產生變異均會提高其對H2O2引起的細胞凋亡之敏感性,而且氧化壓力引起的HO-1產生也被抑制。HO-1的產生受抑制可能與增加氧化壓力引起的細胞凋亡有關。 Parkinson’s disease is one of the most common neurodegenerative diseases. However, the etiology is still not clear. Pink1 is the autosomal recessive gene of the early onset Parkinson’s disease (EOPD), and its mutation counts for much to EOPD. We established five Pink1mutant stable clones in SH-SY5Y: three clones for G309D and two clones for P209A. It was found that more ROS increased in mutant cell lines in response to H2O2 treatment. The apoptotic rate was increased in both G309D and P209A mutant stable clones following H2O2 treatment. Furthermore, Pink1 mutant stable clones had a lower level of dopamine uptake than wild-type after H2O2 treatment. BrdU assay showed that Pink1 mutant stable clones proliferated at a lower rate than that of wild-type. Heme oxygenase 1 (HO-1) usually increases and exerts cell protection when the cells are under oxidative stress. HO-1 increased in wild-type cells following H2O2 treatment. However Pink1 mutant stable clones had a higher basal level of HO-1 and HO-1 can not be induced in response to H2O2 treatment. Using adenovirus infection to overexpress HO-1 and it was found that the H2O2-induced apoptotic rate in Pink1 mutant stable clones can be reversed. These results suggest that the neuroprotective role of Pink1 may have some signaling interaction with HO-1 in SH-SY5Y. Pink1 may be involved in HO-1 induction. On the other hand, it was found that the BDNF mRNA levels are much higher in Pink1 mutant stable clones than in wild-type in resting condition. Following H2O2 treatment, BDNF mRNA was inhibited in both wild-type and Pink1 mutant stable clones. Our results indicate that Pink1 mutant increase the apoptotic sensitivity to H2O2 treatment, and suppression of HO-1 induction but not BDNF production may be involved in the increase of neurotoxicity following oxidative stress. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/26781 |
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顯示於系所單位: | 藥理學科所 |
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