| 摘要: | 依中醫理論，治療神經損傷，宜以益氣活血之中藥為主的精神，選取具有活血化瘀功效的銀杏葉，及其主成份中具有抗凝作用之重要生物活性成分槲皮素，來進行本研究，以確定銀杏葉及其主成分槲皮素是否具有神經保護及增強神經再生的功能。
實驗使用銀杏葉萃取物EGb-761，連續餵食(20 mg/kg/day)小鼠(NMRI mice)一週，使用免疫組織化學染色方法，來觀察小鼠大腦的額葉皮質中，具有調節神經生長和發育之多巴胺受體D1A的表現。另外，使用已被廣泛用在研究細胞分化和神經軸突生長的RSC96細胞株，以槲皮素溶液(0, 0.1, 1.0 and 10.0 μg∕ml)處理兩天，以光學顯微鏡觀察其細胞形態；並以MTT assay測其吸光值，計算細胞相對的存活率；另外，以TUNEL assay與DAPI staining，在螢光顯微鏡下，觀察活細胞及凋亡細胞。此外，以矽膠導管內部充填不同濃度的槲皮素溶液(0, 0.1, 1.0 and 10.0 μg∕ml)，架接在15 mm間隙之截斷的大鼠(SD rats)坐骨神經上，應用組織學和電生理技術，來評估再生神經功能恢復的狀況。
實驗發現，以銀杏葉萃取物EGb-761餵食小鼠，其大腦額葉皮質之組織切片，有較多陽性反應的褐色細胞出現，表示銀杏葉萃取物可上調具有調控神經生長發育基因D1A的表現。另外，使用MTT assay測定細胞相對的存活率，發現槲皮素可顯著地提高許旺氏細胞RSC96的存活和生長。在螢光顯微鏡下觀察，發現幾乎沒有TUNEL-positive的細胞；相反的，只見DAPI-positive的細胞，表示槲皮素並不能誘導許旺氏細胞RSC96凋亡。此外，植入矽膠導管在截斷之大鼠坐骨神經，在組織切片方面顯示，槲皮素組在髓鞘化軸突的數量與密度方面，較對照組顯著增加；在電生理檢測方面，與對照組比較，槲皮素組有比較大的神經傳導速度、比較大的動作電位間期、比較大的振幅、及比較大的CMAP曲線下面積，表示槲皮素能加速神經再生，和神經功能的恢復。
然而，在這次的研究中，在槲皮素濃度為10.0 μg∕ml時，神經再生的成功率，較對照組低，顯示槲皮素對細胞生長有抑制作用，在得到明確結論之前，更進一步關於槲皮素在神經再生方面，其作用機轉的研究是必要的。另外，依據中醫的理論，在未來擴大研究中藥對神經再生的影響，益氣活血中藥是值得優先考慮的。
According to TCM theory, the traditional Chinese medicine is based on Yiqihuoxue(益氣活血) to treat nerve injury. Ginkgo biloba and its most important bio-active ingredient quercetin have been well known one of their function is to remove the thrombus formation in circulation. The goal of this study is to determine the effects of Ginkgo biloba and quercetin, on nerve regeneration.
Using EGb-761(20 mg/kg/day for one week) which is derived from Ginkgo biloba administrated with NMRI mice for a week and investigated whether EGb-761 has function in the regulation of the dopamine receptor D1 expression for nerve growth and development in the frontal cortex of the mouse brain. Moreover, RSC96 cells have been widely used to study cell differentiation and neurite outgrowth. We also applied these cells for study cell morphology, cell survival rate, and nerve growth by difference dose of quercetin (0, 0.1, 1.0 and 10.0 μg∕ml) using MTT and TUNEL assays. To assess quercetin on the recovery of neurological function, we used rat sciatic nerve injury model to fill with quercetin using internal silicone tube and mountings on the truncation of the 15 mm gap.
These results of EGb-761-fed mice show more D1A positive cells in the frontal cortex tissue sections, indicating that ginkgo leaf extract is able to modulate nerve growth and development throung increased D1A expression.
Moreover, quercetin significantly improves the survival and growth of Schwann cells, RSC96 cells, as well as quercetin is able to promote the number and density of the myelin sheath of axons in rats that were implanted silicone tube in the transected sciatic nerves in rats. These results are consist with electrophysiological examine, quercetin-treated rats have relatively large nerve conduction velocity, the larger action potential duration, amplitude, and CMAP area under the curve. Quercetin can not only improve Schwann cells survival rate, but also can speed up the rat sciatic nerve regeneration and functional recovery. However, high concentrations of quercetin have a negative impact in nerve regeneration. It is still not well understood the role of quercetin in the nerve regeneration. Thus, it is necessary to study it in advance in the future. |
