| Abstract: | 本研究的目的是使用體外細胞培養系統來篩選具有抗老化效果的物質。許多文獻指出，與老化有關的半乳醣苷酶 ( Senescence associated β-galactosidase ) 是目前最廣泛使用的老化生物標記，本論文利用此酵素作為細胞老化的指標，建立體外細胞培養系統，並藉以篩選具抗老化活性之中草藥。
在建立老化細胞模式部分，本論文以Fibroblast 3T3 及B16-F0 melanoma 細胞為研究模式，利用X-gal 染色原理，標識老化細胞，實驗結果顯示細胞在0.5% serum 中藍綠色老化標誌較多；染色時間 Overnight 比 4.5 小時明顯；最佳染色 pH 值為6.0；B16-F0 melanoma細胞株染色效果較 3T3佳，形態上也較易觀察。此外在本論文中使用Resveratrol、Polydatin、Trolox和Ascorbic acid來做測試，實驗顯示此系統可做為抗老化活性依據。為探討抗老化與抗氧化及DNA保護的關聯性，實驗上也加入了自由基清除試驗(DPPH˙、ABTS+˙) 、pUC 119 DNA保護試驗以及MTT細胞存活率試驗一起比較。
在篩選中草藥及科學中藥部分：女貞子乙醇萃取液明顯提高老化染色比例，但在DPPH˙及ABTS+˙自由基清除率達77.68%及91.20%。山藥、銀杏葉、山楂及桑枝降低了細胞老化染色的比例，在DPPH˙及ABTS+˙自由基清除試驗，銀杏葉 ( 55.43%, 77.51% )、山楂 ( 41.59%, 55.26% ) 及桑枝 ( 33.77%, 84.29% ) 具有清除自由基能力，而山藥清除能力較差，只有 7.65% 和 15.51%。骨碎補 ( 71.38%, 87.67% )、雞血藤 ( 70.36%, 88.00% ) DPPH˙ 及ABTS+˙自由基清除率佳，也顯示有 DNA 保護能力，但老化試驗中並無降低細胞老化染色比例。
系統顯示具有抗氧化或是 DNA 保護力之中草藥或科學中藥不一定具有抗老化的能力，具有抗老化效果之中草藥或科學中藥也不一定具有抗氧化或是DNA 保護能力，這個結果顯示老化過程極為複雜，單獨檢測抗氧化或DNA保護試驗可能無法反應抗老化之結果，因此需要更多實驗才能深入探討抗老化之機制。
The purpose of this study is using in vitro cell culture systems for screening substances with anti-aging effect. Many studies showed that the Senescence-associated β-galactosidase is currently the most widely used aging biomarkers, in this study , this enzyme was used as an indicator of cell-aging.
In the part of establishment of aging cell model, we used Fibroblast 3T3 and B16-F0 melanoma cell line. X-gal staining was performed. Results showed that under 0.5% serum senescence marker is more than others, staining overnight is better than four hours, senescence marker is much more when under pH 6.0 rather than under pH 7.4, the posotive staining on B16-F0 cell lines was more and easier in observation than in 3T3 cell lines because of the cell morphology. However, UVA irradiation did not significent increased the proportion of cellular senescence staining. In addition, Resveratrol, Polydatin, Trolox, and Ascorbic acid were used in this study to test the model. To investigate the association of anti – aging, antioxidant and DNA protection, we also performed DPPH˙scavenging assay, ABTS+˙scavenging assay, pUC 119 plasmid DNA protection assay and MTT assay.
In the part of screening Chinese herbal medicine and scientific medicine : Privet Fructus EtOH extract significantly increased the proportion of cellular senescence staining, but the scavenging ability in DPPH˙ and ABTS+˙ free radicals was 77.68% and 91.20%. Dioscorae Rhizoma, Ginkgo biloba, Crataegus, and Mori Ramulus reduced the proportion of cellular senescence staining, in DPPH˙ and ABTS+˙ free radicals scavenging assay, Ginkgo biloba (55.43%, 77.51%), Crataegus (41.59%, 55.26%) and Mori Ramulus (33.77%, 84.29%) had free radical scavenging capacity while Dioscorae Rhizoma had poor scavenging ability, it was only 7.65% and 15.51%. Drynariae Rhizoma (71.38%, 87.67%) and Millettia dielsiana (70.36%, 88.00%) were good at DPPH˙ and ABTS+˙ free radicals scavenging and also showed DNA protection but does not reduce the proportion of cellular senescence staining.
This system displayed the capacity of antioxidant or DNA protection ability of Chinese herbal medicine or scientific medicine was not necessarily equal to anti-aging ability, anti-aging effects of Chinese herbal medicine or scientific medicine was not necessarily have antioxidant or DNA protection ability, these results indicate that the aging process is extremely complex, the antioxidant or DNA protection ability could not reflect the results of the anti-aging effects, therefore, more experiments are needed to further explore the mechanisms of anti-aging. |
