| Summary: | 碩士 === 國立臺灣師範大學 === 生命科學研究所 === 102 === Neurodegenerative disorders, such as Alzheimer's disease and Parkinson's disease cause irreversible neuronal death that impairs brain functions. The discovery of neural stem cells in the adult brain provides possibilities that we can induce these cells to differentiate into neurons lost in neurodegenerative disorder. To screen for potential neurogenic compounds, we started with P19 cell cultures. P19 cells are pluripotent stem cell-like cells, which can be induced to differentiate into neural and muscle cells by drug treatments or transfected with neural basic helix-loop-helix (bHLH) transcription factors. In the first round of screening, P19 cells were cultured with various Chinese herbal extracts of different concentrations provided by Sun Ten Pharmaceutical or Industrial Technology Research Institute for 3 days. We found that 17% of screened Chinese herbal extracts induced neuronal differentiation in this culture condition. In the second round of screening, P19 cells were cultured to form aggregates with retinoic acid (RA), a known molecule to induce neuronal differentiation, first and then cultured as monolayer with those herbal extracts identified from the first round of screening. After the second round of screening, we found that 5.1% of screened Chinese herbal extracts, including NH002, NH003, NH005 (from Sun Ten Pharmaceutical) and drug 379 (from Industrial Technology Research Institute) promoted neuronal differentiation. To further confirm their neurogenic effect, neural stem cells isolated from the subventricular zone of the postnatal day 7 mouse brain were cultured to form neurospheres. Dissociated neurosphere cells were treated with Chinese herbal extracts from the second round of screening. We found that NH003 and NH005 promote neuronal differentiation in neural stem cells. Furthermore, we found that NH003 and NH005 increased the expression of β-catenin, the key effector protein of the Wnt signaling known for its role in neuronal differentiation. To examine whether β-catenin mediates the neurogenic effect of NH003 and NH005, P19 cells were transfected with shRNA against β-catenin and cultured with NH003 or NH005. We found that knockdown of β-catenin blocked neuronal differentiation in NH003 and NH005 culture. To make sure the neurogenic effect was not due to increased cell proliferation, we added BrdU two hours before fixation to label proliferating cells in NH003 or NH005 treatments. BrdU-positive cell numbers were not changed during NH003 or NH005 group. Therefore, NH003 and NH005 have no effect on cell proliferation. Taken together, our study suggests that NH003 and NH005 promote neuronal differentiation through Wnt/β-catenin pathway without affecting proliferation. Lastly, we found that ginkgolid A could be one of the effective components from NH003 (ginkgo biloba) to induce neuronal differentiation. In conclusion, Chinese herbal medicines could be potential treatments for neurodegenerative disorders.
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