1.本論文所設定之定性與定量分析模式，均可廣泛運用於神經細胞生長促進劑之開 發研究。本實驗所初步篩選之中藥可比照此方法進行進一步的篩選及開發研究。
2.未來預期繼續之工作
單複方或電針各組選出可使細胞存活且高冷光反應的藥物方法者進一步進行動物 動物記憶試驗( 如走迷宮，雛雞實驗等)進一步求證及篩選。
3.未來展望
此系統可用於尋找與神經生長因子（NGF）作用類似之藥物或治療（如電磁效應）
以尋求及評估促進神經生長分化之各種治療方法（圖 6.1,6.2）。我們的實驗室目 前擁有 500 餘種甲醇萃取之中藥庫(附錄二)，未來一年將擴充超過 500 種以上,利 用以 SH-SY5Y/AP-1/LUC 重組細胞中 Luciferase 產生之冷光強度為 AP-1 活性指 標作為神經細胞生長之測定標準，可以建立尋找及評估誘發神經細胞生長分化藥 物的快速搜尋平台系統。未來經此系統分析有效的中醫藥療法可考慮進一步納入 動物實驗或臨床實驗加以求證，可縮短減少開發流程時間及成本。
圖 6.1 未來實驗流程 Fos Jun
AP-1
單方,複方 or 電針 or NGF
篩選出冷光強者進一步進行動物實驗
AP-1 / Luc MTT test
圖 6.2 未來實驗步驟
單 方 ,複 方 or 電 針 or NGF
x 2
MTT test AP-1 / Luc
選出可使細胞存活且高冷光反應 的藥物方法
篩 選 出 冷 光 強 者 進 一 步 進 行 動 物 實 驗
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附錄一 本實驗選用的藥物
•中藥成份：Baicalein, Baicalin, Formononetin, Ginkgolide A, Ginkgolide B
•西藥:Valproic acid (VPA)
•複方:加味逍遙散
Ginkgoflavones: Ginkgolide A, Ginkgolide B
附錄二 本實驗室目前收集的中藥庫的收集
Study on the Molecular Mechanism of the Chinese Medicinal Herbs in the Promotion of Neurite Growth
Chang-Hsien Lee
Major professor: Jaung-Geng Lin
Institute of Chinese Medical Science, China Medical college Taichung, Taiwan, ROC
Background and Goal
We began to observe the human neuroblasma cell ( SH-SY5Y ) in this experiment, and used a drug ( Valproic acid ) to be a positive control group.
Valproic acid ( VPA ) is a proofed stimulate substance to improve nerve growthing in magazine. We want to build a experiment model to develop and find new neutrophic factors ( NTFs,a substance which can promote nerve growth) soon.
Material and Method
SH-SY5Y cell is a human neuroblasma cell which possess many nerve properties to be a nerve cell model . SH-SY5Y cell is good used to be a nerve growth model to invest NGF ( nerve growth factor) or other NTFs .
In this experiment ,we used sixty-four species of Chinese herb extracts
to select best twenty NTFs . In the best twenty NTFs ,we find some flavon ingredients from Chinese herbs have good effect in prompting nerve growth.
We followed to select twenty-six c ommon flavon ingredients in advance experiment. The best three ingredients are Rutin, Hyperin, Gingkgolid B ( GB ). GB has especially effect to prompt SH-SY5Y cell growth among this three ingredients,so we select Gingkgolid B in following experiment.
We added Gingkgolid A, B and VPA individually in a different dose growth medium to SH-SY5Y cell and observe the cell activity by MTT assay test.
In quantitative measurement experiment, we import the digital camera photos into computer to calculate the SH-SY5Y cell axon lengths in different petri dish and analysis by Image-pro Lite software.
NGF is the first found and known substance as a NTFs . Many scholars indicate it prompt nerve growth through ERK and AP-1 pathway. AP-1 is a early immediate response protein to regulate the gene activity to control cell differentiation and proliferation. We investigate GB molecule mechanism by western blot electrophoresis method to
explore GB prompt nerve growth by AP-1 pathway or other pathway.
Result
1. Both GA and GB are the substance to prompt nerve growth.
2. In western blot electrophoresis method , we find:
<1>In VPA group (positive control group), ERK~P increase in 30 mins . The result is same to the experiment result in previous papers . <2> In GB group (experiment group), ERK~P does not increase in 30 mins .
It decrease graduate. It is possible, GB prompt nerve growth by active other pathway substance first, and ERK~p is involved secondly.
3. The MTT assay result show: GA, GB and VPA do not possess acute poison (in 48 hours).
Conclusion
First, we can use the qualitative and quantitative measurement experiment method to elevates the neutrophil effect of new NTFs widely.
Secondly, w e can invest the best twenty herbs which we screen from sixty-four Chinese herbs and ingredients by the same method. In the future, we can confirm the neutrophil effect by animal or clinical experiment in advance in the future.