骨碎補對脂源性間葉幹細胞分化骨質效應之研究

Effects of Drynariae Rhizoma on Bone Differentiated by Adipose Mesenchymal Stem Cell

10.6832/KMU.2007.00147

郭明和

脂源性間葉幹細胞 ； 骨質誘發 ； 骨碎補 ； Adipose mesenchymal stem cell ； differentiated osteogensis ； Drynariae Rhizoma

高雄醫學大學醫藥暨應用化學研究所碩士在職專班學位論文

2007年

碩士

蔡錦蓮

繁體中文

由於國人壽命日趨年長，全球逐漸步入老年化世界。伴隨著年齡增長，骨質疏鬆症已被認為是影響大眾健康主要的疾病之一。因此，本研究利用脂源性間葉幹細胞可以分化成硬骨的特性，加入骨碎補，探討是否對骨質的形成有所助益。 本研究將人類脂肪萃取出間葉幹細胞，研究結果，MTT試驗，骨碎補在16天時100 ppm仍不影響生長速率，但培養20天試驗時，大於20 ppm即會影響細胞生長，所以本研究以10及15 ppm做探討濃度。骨質誘發試驗，骨碎補15 ppm誘發培養十日時，細胞外觀沒有變化，鈣化節點染色和細胞含鈣濃度實驗組和控制組並無顯著差異。誘發培養14日時，發現含骨碎補的骨質誘發實驗組及陽性控制組之培養液中鈣濃度比控制組明顯的減少。含骨碎補的骨質誘發實驗組在誘發培養二十八日後發現鹼性磷酸酶、鈣化節點染色和細胞含鈣濃度（實驗組鈣濃度為17.3 ppm；陽性控制組鈣濃度為4.9 ppm）和陽性控制組有顯著差異。結果顯示，加入骨碎補可促進增加脂源性間葉幹細胞分化成骨質細胞。 骨形成相關蛋白研究顯示，在第10天，鹼性磷酸酶及表現非常強烈，但其他的Osteocalcin，Osteoprotegerin及Osteopontin的表現不如鹼性磷酸酶蛋白表現強烈，只有顯示骨碎補加藥組比陽性控制組表現較強。而Integrin β則未在骨誘發過程有表現。 本研究可證明骨碎補多酚可直接促進脂源性成體間葉幹細胞分化成骨質細胞，增加功能性的骨細胞。

As the older population increases, the global has changed gradually into elder society. Due to the growth of age, the incidence of osteoporotic fractures is expected to dramatically rise during the next decades. Adipose tissue has several clear advantages as an initial material for harvesting stem cell, as it is abundant and relatively easy to procedure. Therefore, the objective of this study discusses whether it is helpful for formation of osteoid after adding Drynariae Rhizoma extract(DR). The method is to use the characteristics of being differentiated osteogenesis by adipose mesenchymal stem cell. The significant concentrations of Drynariae Rhizoma are below 20 ppm by MTT assay respectively. The result of alkaline phosphatase atain, von kossa stain and calcium concentration accumulated in the cells is not significant differently by utilising Drynariae Rhizoma incubated for 16 days between the experiment group and the negative control group. Positive staining with von Kossa staining (black) has revealed that these deposits are indeed calcified ECM. Quantitative measurement of calcium using a flame atomic absorption spectrophotometer showed that the differentiation induction plates had 17-fold increases in calcium content compared to the control. The measurement of calcium in the medium also confirms that calcium contents in the medium were depleted in plates containing differentiated AD-MSCs but not in the control plates at the end of each incubation period. The induction of osteogenesis in postivie control and D.R. treated were extensive.As can be observed, more than 80% of the cells were covered with calcified ECM. In differentiated osteogensis with Drynariae Rhizoma, there are significant differences among alkaline phosphatase stain, von kossa stain and calcium accumulation in the cells than the negaive control group after 28 days incubated (calcium concentration in differentiated osteogensis with Drynariae Rhizoma is 17.3 ppm ; in differentiated osteogensis is 4.9 ppm). According the study for formation of osteoid, in tenth day, the alkaline phosphatase stain in better than Ostecocalcin, Osteoprotegerin, and Osteoptin. Only D.R. better than nevaive control group. There is no effect of Integrin β in the differentiated osteogensis. This study is showing that D.R. can improve the differentiated osteogensis by adipose mesenchymal stem cell, increase the functional bone cells.

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