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  • 學位論文

槲黃素對造骨細胞活性之影響

The Biological Effects of Quercetin on the Osteoblast

指導教授 : 翁清松
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摘要


摘 要 造骨細胞主宰骨質形成的調控,在發炎反應中造骨細胞(osteoblast)的增殖、分化及與蝕骨細胞間的作用是骨質形成中很重要的步驟,一旦造骨細胞分化不足或受活化的蝕骨細胞數目過多,骨質的生成與骨質密度將會降低,嚴重地影響骨骼生長修復的情形。 本研究利用天然物-槐花經溶劑萃取成黃酮類萃取物槲黃素(Quercetin),於體外的細胞培養實驗模式觀察其對造骨細胞(MC3T3-E1)活性的作用,再利用脂多醣誘導造骨細胞分泌發炎調節因子(NO2-/NO3-、IL-6、IL-1β)模式,分別加入槲黃素與維生素C共同作用,探討對發炎調節因子影響,期望釐清槲黃素與發炎調節因子之間的作用,將來對於治療骨骼疾病的天然藥物方面有所貢獻。 本實驗分為兩階段,首先先測定槲黃素(0.1~100μM)對造骨細胞存活率(12、24、48與72小時)及造骨細胞分泌鹼性磷酸酵素(48小時)的影響。再利用特定濃度的脂多醣(5μg/ml)誘導造骨細胞分泌發炎調節因子(NO2-/NO3-、IL-6、IL-1β)的模式,分別加入槲黃素與維生素C共同作用48小時,探討對發炎調節因子的影響。 研究結果發現,在細胞存活率測試(MTT ASSAY)顯示從天然物萃取出之槲黃素,在低濃度的劑量(0.1~1μM)下加入造骨細胞中培養72小時後有促進造骨細胞增生的效果,以1μM的濃度其增生率最高;而由鹼性磷酸酵素(ALP)的含量測定,添加0.1μM~5μM的槲黃素至細胞中共同培養48小時後,ALP含量大約增加了20~40%,以濃度為1μM的效果最顯著。因此,本研究發現從槐花萃取出之槲黃素在濃度為0.1~1μM下可促進造骨細胞增生與分化。 第二階段探討槲黃素對造骨細胞分泌發炎調節因子的影響,研究結果顯示,濃度0.1~5μM的槲黃素均有抑制造骨細胞分泌NO2-/NO3-、IL-6的現象,分別抑制40﹪與30﹪的生成量。而添加脂多醣(5μg/ml)誘導造骨細胞後發現,0.1~5μM的槲黃素均可抑制由脂多醣誘導造骨細胞所分泌的NO2-/NO3-與IL-6。最多可抑制兩者的產生達40﹪與50﹪;而濃度0.5~5μM的槲黃素可抑制IL-1β的產生達32﹪。對照加入維生素C(100μM)至脂多醣活化的造骨細胞共同培養作用下,發現維生素C也可抑制脂多醣誘導造骨細胞所分泌的發炎調節因子產生,但此抑制的效果較槲黃素差。

並列摘要


Abstract Osteoblasts are important cells for the formation of bone tissue in skeletion. Decreased function or decreased number of osteoblasts may result in bone loss. Under inflammatory conditions, the proliferation and differentiation of osteoblasts and the relationships between osteoblasts and osteoclasts are responsible for renewing the skeleton while maintaining its anatomical and structural integrity. The bone resorption process is closely dependent on the active osteoclasts. If the number of activated osteoclasts increase and the number of osteoblasts decrease too much, bone integrity will not be conservative. Hence, protecting the viability of osteoblasts may reduce the risk for osteoporosis and associated fratures. Flavonids are major components of natural products. The aim of this study was to investigate how quercetin, which is one of the components extracted from Sophora japonica L., affected the activity of osteoblasts. Then, this study was conducted to determine how quercetin or vitamin C affect the production of nitrite/nitrate (NO2-/NO3-), interleukin-6 (IL-6) and interleukin-1β (IL-1β) which were induced by Lipopolysaccharide (LPS) treatment, respectively. To study the effects of quercetin on the osteoblasts, the crude extract was extracted from Sophora japonica L.. The effects of quercetin extracts on proliferation and differentiation of osteoblast MC3T3-E1 were first evaluated by the assays of MTT (3-(4, 5-dimethylythiazol-2-yl)-2, 5-diphyenyl tetrazoloum bromide) (12, 24, 48, 72 hours) and alkaline phosphatase activity (48 hours), respectively. And then, we used LPS to determine how quercetin or vitamin C affected the productions of NO2-/NO3-, IL-6 and IL-1β which were induced by LPS treatment (48 hours), respectively. The results showed that a significant increase by 10% in MC3T3-E1 cell proliferation was found with 72 hours treatment of quercetin at 1μM as compared to the control group, and a significant increase by 20~40% in cell differentiation was found with 48 hours treatment of quercetin at 0.1~5μM. Also, we found that quercetin significantly increased osteoblast proliferation and differentiation. Moreover, our study indicated that quercetin (0.1~5μM) showed the significant inhibition under normal condition or on LPS-induced NO2-/NO3-, IL-6 and IL-1β (48 hours). Quercetin inhibited LPS-induced the productions of NO2-/NO3-, IL-6 and IL-1β. The production of NO2-/NO3- was decreased at most to 40%. The productions of IL-6 and IL-1β were decreased at most to 40% and 32%, respectively. We also found that treatment of osteoblasts with Vitamin C (100μM) followed by LPS treatment could inhibit LPS, which induced NO2-/NO3-, IL-6, IL-1β productions.

並列關鍵字

Vitamin C IL-6 Osteoblast Cytokine NO2-/NO3- Quercetin Lipopolysaccharide IL-1β

參考文獻


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