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研究生(外文):TaoLin Cheng
論文名稱(外文):Protective Effect of 1α,25-Dihydroxyvitamin D3 in Primary Cultured Neuronal Cells
指導教授:王 昀吳劍男
指導教授(外文):Wang YunWu Jian Nan
外文關鍵詞:125-dihydroxyvitamin D3N-methyl-D-aspartatehydrogen peroxideprimary cultured neuronal cellsneuronal protection
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最近的文獻報告顯示1,25-dihydroxyvitamin D3{1,25-(OH)2 D3}可以刺激神經滋養因子,如:nerve growth factor ( NGF )、glial cell line-derived neurotrophic factor ( GDNF )、neurotrophin-3 ( NT-3 )的基因表現。而這些神經滋養因子皆具有神經滋養及神經細胞保護作用。而1,25-(OH)2 D3是否具有相類似神經滋養或神經保護作用目前不清楚。
在本研究中,我們利用鼠胚胎腦進行細胞培養。我們發現在大腦皮質細胞或中腦培養細胞,單獨給予高濃度( 10-6 M )1,25-(OH)2 D3可造成毒性反應。由MTT assay可以造成看出細胞10-6 M下明顯死亡。而低濃度( 10-9 ~ 10-12 M )的1,25-(OH)2 D3下細胞並則沒有毒性作用。另外,利用鈣離子螢光法測量神經細胞鈣離子濃度,發現高濃度( 10-7與10-6 M ) 1,25-(OH)2 D3可造成細胞內鈣離子濃度上昇,但是低濃度1,25-(OH)2 D3處理後並不明顯。因此,高濃度1,25-(OH)2 D3所產生的細胞死亡可能是因為增加細胞中鈣離子所造成的。此結果也顯示,1,25-(OH)2 D3對培養細胞不具有neurotrophic之作用。
加入10-3 M NMDA二十四小時後,從大腦皮質細胞外觀以及免疫螢光染色觀察可發現神經細胞細胞萎縮,神經軸消失。利用LDH測量細胞退化程度,發現NMDA可以增加LDH活性。如事前加入10-9 M或10-10 M 1,25-(OH)2 D3能明顯降低NMDA對LDH的作用。這些結果顯示1,25-(OH)2 D3能保護大腦皮質神經細胞對抗NMDA所產生的毒性反應
初級培養大腦皮質細胞或中腦培養細胞,在加入10-3 M與 10-4 M H2O2二十四小時後,從細胞外觀以及免疫螢光染色觀察神經細胞,發現H2O2產生細胞毒性造成大腦皮質細胞或中腦神經細胞死亡。利用LDH測量法及MTT測量細胞退化程度,發現LDH活性增加,MTT吸光值明顯下降。事前加入10-10 M 1,25-(OH)2 D3能明顯降低10-4 M H2O2所引起的LDH活性,並增加MTT吸光值。這些結果顯示(OH)2 D3能保護神經細胞能對抗H2O2所產生的大腦皮質細胞或中腦神經毒性。

Recent studies have indicated that 1,25-dihydroxyvitamin D3 (or D3) increases the expression of various trophic factors, such as nerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF), neurotrophin-3 (NT-3). These trophic factors have been reported previously to induce neurotrophic or neuroprotective effects. It is still not clear if D3 can produce similar effects in neuronal culture.
In this study, we used primary cell culture obtained from cerebral cortex or ventromesencephalon (VM) of rat embryos. We found that application of D3, at 10-6 M concentration, induces cell death, as detected by MTT staining, in cortical or VM cell culture. D3, at much lower concentration (10-9 M to 10 -12 M), did not decrease MTT activity. Furthermore, we found that D3, at 10-7 and 10-6 M, but not 10-9 M to 10-12 M concentration, increases intracellular Ca++ levels as determined by Fura-2 fluorescent. These data may suggest that calcium is involved in D3-induced neurotoxicity at high D3 concentration. Since D3 does not increase MTT activity, it may also suggest that D3 does not have trophic effects to the culture cells.
We found that the addition of 10-3 M NMDA or 10-3 M - 10-4 M H2O2 for 24 hours increases LDH levels and decreases cell survival in cerebral cortex or ventromesencephalon (VM) cells. However, pre-treatment with 10-9 M or 10-10 M D3 significantly decreases these NMDA or H2O2 -mediated neurotoxicity. These data suggest that D3 has neuroprotective effect against NMDA and H2O2 in cerebral cortex or ventromesencephalon (VM) cells.
In conclusion, our experiment demonstrated that pre-treatment of D3 decreases NMDA and H2O2-induced neurotoxicity. Such effects may be secondary to the upregulation of neurotrophic factor in the culture cells.


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