| 摘要: | 本研究主要針對台灣產民間藥對於ICR小鼠骨髓細胞系中成熟蝕骨細胞(osteoclast-like multinucleated cells；MNCs)的影響，並藉由酵素連結免疫吸附分析法（enzyme-linked immunosorbent assay；ELISA）來了解這些台灣產民間藥對於蝕骨細胞分化成熟過程中的調控因子(IL-1α, IL-6, TNF-α, PGE2)有何影響。本研究所採用的台灣產民間藥分別為：冇骨消 (Sambucus formosana Nakai )；刺仔頭 (Acacia farnesiana (L.) Willd.)；細本山葡萄 (Vitis thunbergii Sieb. & Zucc. var. adstricta (Hance) Gahnep)；肺形草 (Tripterospermum taiwanense (Masamune) Satake)及黃金桂(Cudrania cochinchinensis (Lour.) Kudo et Masam. var. gerontogea (Sieb. & Zucc.) Kudo et Masam.)。
在體外研究模式中，以1a, 25-(OH)2D3誘發蝕骨細胞的生長，同時分別加入5µg/ml、50µg/ml、500 µg/ml三種濃度之上述藥物抽出物，培養七天後，以TRAP（tartrate-resistant acid phosphatase）染色法，觀察這些台灣產民間藥對於蝕骨細胞的生長是否具抑制性。結果發現，當藥物濃度為500 µg/ml時，加入冇骨消、刺仔頭、細本山葡萄、肺形草、黃金桂水抽出物、黃金桂酒精抽出物等組，均可明顯抑制成熟蝕骨細胞的生長；而在50µg/ml濃度時，除刺仔頭組不具顯著差異外，其餘均可明顯抑制成熟蝕骨細胞的生長。且發現50µg/ml濃度之藥物抽出物，對蝕骨細胞之抑制為可逆現象。接著以MTT偵測技術分析細胞的存活率，在成骨細胞方面，發現上述藥物抽出物對於成骨細胞不具毒性作用，反而在冇骨消（500µg/ml）、肺形草（5、50、500 µg/ml）、黃金桂（5、50、500µg/ml）等加藥組中，發現成骨細胞具增生作用。在蝕骨細胞方面，濃度為50、500 µg/ml之上述五種藥物抽出物，均有明顯降低蝕骨細胞生長，但500 µg/ml的冇骨消、肺形草、黃金桂酒精抽出物組，對細胞具有毒性。
雌激素在蝕骨細胞的分化及活化過程中，為一重要調控因子，近年來發現蝕骨細胞(osteoclast：OC)及成骨細胞（osteoblast：OB）均含有雌激素之接受器。因此以去除卵巢老鼠之骨髓細胞做體外培養，分別加入上述藥物抽出物及雌激素（17b-Estradiol），觀察對成熟蝕骨細胞的影響性。結果發現此五種藥物抽出物，可降低因雌激素缺乏所導致之蝕骨細胞活化作用，對其生長具抑制作用。再來以17 b-Estradiol kit測得17 b-Estradiol的含量，在第四天偵測，發現50、500µg/ml濃度之各加藥組中，17 b-Estradiol量較控制組（1 a,25-(OH)2D3）明顯增高許多。這說明了，此五種台灣民間藥抽出物，可能具有類似17 b-Estradiol結構之植物性雌激素。另一方面，和蝕骨作用有關的細胞激素（如：IL-1 a, IL-6, TNF- a, PGE2）已經被證實可促進成熟蝕骨細胞的形成，進而導致骨質流失。為了要了解這些台灣產民間藥是否會對這些細胞激素有所影響，我們以ELISA的方法，測骨髓細胞培養系中細胞懸浮液之IL-1 a, IL-6, TNF- a, PGE2濃度，觀察這些台灣產民間藥之所以能抑制蝕骨細胞生長，是否藉由降低蝕骨作用有關的細胞激素而達到抑制的作用。結果發現50µg/ml、500µg/ml濃度之五種台灣民間藥，可藉由降低IL-1 a, IL-6, TNF- a而達到成熟蝕骨細胞抑制的作用，然而本實驗中PGE2並無顯著差異。從上述結果推論，本研究所採用之五種台灣民間藥，可能藉由降低蝕骨作用相關的細胞激素（IL-1 a, IL-6, TNF- a），進而達到抑制成熟蝕骨細胞數。另外這些台灣民間藥可能含有一些植物性雌激素，經由這些植物性雌激素的作用，而達到抑制成熟蝕骨細胞數目。
The aim of this study is to investigate how several Taiwan folk medicines affect the development of Osteoclast-like Multinucleated cells (MNCs) in tibia bone marrow of ICR male mice. This study was also conducted to determine how cytokines (i.e. IL-1, IL-6, TNF-α, PGE2) affect the formation of MNCs in tibia bone marrow of the ICR male mice. Several Taiwan folk medicines, such as Sambucus formosana Nakai (Sf )；Acacia farnesiana (L.) Willd. (Af)；Vitis thunbergii Sieb. & Zucc. var. adstricta (Hance) Gahnep (Vt)；Triyerospermum taiwanense (Masamune) Satake (Tt)； Cudrania cochinchinensis (Lour.) Kudo et Masam. var. gerontogea (Sieb. & Zucc.) Kudo et Masam. (Cc) were chosen for this research. First of the study, bone marrow cells were flushed from the male ICR mice (6-weeks-old) tibia bone. 1a,25-(OH)2 D3 and various concentrations (5, 50, 500 µg/ml) of each Taiwan folk medicine were added to the plates. At day 8, these MNCs were stained with tartrate-resistant acid phosphatase（TRAP）. Results showed that MNCs were increased in the 1 a,25-(OH)2D3-treated group (389.50±38.41；p<0.01), which was statistically significant. The other groups with 1 a,25-(OH)2D3 and 500µg/ml of Sf（9.56 ±2.33；p<0.01）, Af（16.83 ±5.75；p<0.01）, Vt（50.29 ±6.33；p<0.05）, Tt（24.57 ±8.10；p<0.01）, Cc-EtOH（60.71 ± 10.3；p<0.05）, Cc-EtOH（24.40 ± 8.91；p<0.05）significantly inhibited the MNCs formation when compared to 1α, 25-(OH)2D3-treated group. The group on 50µg/ml concentration of Sf（94.50±8.70；p<0.05）, Af（184.62±22.11；p<0.05）, Vt（161.36±12.3；p<0.05）, Tt（75.30 ±9.00；p<0.01）, Cc-H2O（100.43± 11.67；p<0.05）, Cc-EtOH（103.00±12.4；p<0.05）significantly inhibited the MNCs formation when compared to 1α, 25-(OH)2D3 -treated group. These results showed dose-dependence relationship. We also investigated the cytotoxic effect of these Taiwan folk medicines（5, 50, 500µg/ml）in osteoblastic cells and osteoclast cells using MTT assay. Sf, Tt, Cc showed cytotoxity on 500µg/ml concentration. As a result, we selected a suitable concentration for several Taiwan folk medicines , such as 50µg/ml of Af, Tt, Cc-EtOH. 50, 500 µg/ml of Vt, Cc-EtOH. Finally, we studied how these Taiwan folk medicines affected the levels of IL-1 a, TNF- a, IL-6, PGE2 in regulating the differentiation and activation of osteoclast. We found that these Taiwan folk medicines with their inhibition of MNCs in tibia bone marrow cells could probably be regulated by the alteration of IL-1 a, TNF- a and IL-6. In addition, 17b-estradiol like substances were detected by ELISA kit. This suggests that these Taiwan folk medicines could have dedicated their phytoestrogens to the tibia bone marrow cells. |
