臺灣博碩士論文加值系統

維他命B1是一個參與在許多有氧及氧化代謝的重要輔酶，哺乳動物並無法自行合成，故缺乏時常造成病症。本研究以腦皮層神經細胞為模型，利用維他命B1相似物amprolium來抑制細胞吸收維他命B1，進而探討探討維他命B1缺乏對細胞之影響。藉由直接的細胞型態、細胞計數，觀察到維他命B1缺乏使得腦皮層神經細胞缺失，而由其細胞週期及TUNEL實驗等分析證明，細胞的減少在於細胞進行程序性凋亡，並伴隨著caspase 3、8、9活性的上升及下游PARP蛋白之切割。此外，細胞內活性氧分子含量（ROS）也會隨著維他命B1缺乏而上升，但活性氧分子抑制劑卻無法挽救細胞之死亡，故推測維他命B1缺乏所造成的細胞死亡並非由ROS所主控。另一方面， ERK及JNK的磷酸化皆有降低的趨勢。以ERK及JNK活性的抑制劑：PD 98059、U 0126以及SP 600125處理細胞，觀察到不論是否處於維他命B1缺乏的狀態，ERK活性之抑制劑並不會影響細胞的存活；相對的，JNK活性之抑制劑卻明顯造成腦皮層神經細胞嚴重死亡，這意味著JNK活性與細胞存活密切相關。在維他命B1缺乏的情況下，p53或是其Ser15蛋白磷酸化的量皆會上升，而負責運輸維他命B1的thiamine transporter （THTR-1）的mRNA量亦有增加。但上述分子的變化與維他命B1缺乏造成死亡的關聯，仍有待進一步的研究。

Vitamin B1 (Thiamine) plays a critical role in energy and oxidative metabolism, its deficiency can result in abnormal oxidative degradation of nutrients. By treatment of cells with the thiamine antagonist amprolium as a means of inducing thiamine deficiency, we showed that thiamine deficiency elicited apoptosis in cultured cortical neuronal cells. The apoptosis was accompanied by the activation of caspases 3, 8, and 9 and a two-fold increase of reactive oxygen species (ROS) was detected. Treatment of cells with ROS inhibitors, NAC and vitamin C, did not prevent the amprolium induced death of neuronal cells. Western blot analysis using antibodies specific for phosphorylated MAP kinases demonstrated that amprolium treatment led to a decrease in the level of phosphorylated JNK and ERK. Exposure of cells to the JNK inhibitor SP600125, but not ERK inhibitor PD98059 and U0126, resulted in severe neuron death, suggesting that the activity of JNK is important to the survival of cortex neurons. The expression of p53 and its Ser-15 phosphorylation form was increased; and RT-PCR analysis indicated that the level of THTR-1, a p53 target gene, was upregulated. Mechanisms underlying the deficiency of vitamin B1 induced apoptosis remain to be determined.

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