骨頭疾病造成了現今社會大眾許多患者的困擾。前人研究僅探討龜鹿二仙膠(由鹿角、龜板膠、枸杞和人參組成)在骨頭疾病上的療效。如果其促骨生成的主成分可被檢驗出來，則作用機制可被進一步的研究。根據古籍，龜板膠為最有可能有促骨生成效用之成分。然而，到目前為止並無研究探討龜板膠之骨生成效果。而本研究發現單方龜板膠可以促進離體及活體模式中的骨生成的作用並促進人類骨母細胞MG-63增生、ALP及礦化活性。此外，我們發現龜板膠可以活化Akt和ERK訊息路徑且顯著增加骨鈣化相關基因如Runx2, 骨鈣素和骨橋蛋白及其蛋白質的表現。我們利用斑馬魚及大鼠活體模式確認龜板膠的促骨活性。動物實驗結果顯示斑馬魚在給予龜板膠72小時後，具有促進骨生成的作用。而口服1.25 g/kg龜板膠則可顯著增進大鼠顱骨缺損的復原並伴隨血清ALP的增加。總結，龜板膠提供給需要骨再生治療的病人一個可能的治療選擇。

Many patients suffer from bone-related diseases. Previous studies have investigated the use of Gui Lu Er Xian Jiao, which is composed of four different ingredients including antlers, colla plastri testudinis, wolfberry, and ginseng, to treat bone diseases. If the effective component of Gui Lu Er Xian Jiao can be identified, we can further study the underlying mechanisms. According to ancient scriptures, the component most likely has a bone-forming effect is colla plastri testudinis (CPT). However, there is no scientific date to support this hypothesis. In the present study, we found that only one prescription of CPT promotes osteogenesis both in vivo and in vitro, and could promote the proliferation of human osteoblast cells, MG-63 and the activity of ALP, and mineralization. In addition, we found that CPT could activate the Akt and ERK signaling pathways and significantly increase the expression of ossification-related genes, such as Runx2, osteocalcin, and osteopontin, and their corresponding proteins. Zebrafish and rat models were used to confirm the osteogenic effects of CPT in vivo. Animal studies revealed that osteogenesis was activated when zebrafish were immersed in CPT for 72 h. An oral dose of 1.25 g/kg CPT could significantly improve skull defects in rats, accompanied by an increase in serum ALP levels. In conclusion, CPT provides a promising therapeutic option for patients requiring bone regenerative treatments.

論文審定書. i
摘要 ii
Abstract iii
目錄. iv
表目錄. ix
中英文對照及縮寫表 x
第壹章、前言 1
骨代謝在硬骨組織的角色 1
骨形成相關流行病學 1
齒科疾病及骨質新生 2
骨質新生機轉 3
骨質新生的藥物開發 7
中草藥於老藥新用之優勢 8
龜鹿二仙膠介紹 9
研究動機 11
第贰章、材料與方法 12
CPT 製備 12
細胞培養 12
CPT 滅菌 12
細胞存活率 12
ALP 活性測試(Alkaline phosphatase activity) . 13
細胞蛋白質定量 13
SDS-polyacrylamide gel 製備 14
電泳 14
電泳轉印(transfer blot) 14
抗體接合 15
即時定量聚合酶連鎖反應 15
v
礦化能力試驗 16
種魚飼養 17
胚胎取得 17
實驗仔魚飼養 17
藥物安全濃度篩選 17
斑馬魚鈣黃綠素染色 18
顱骨缺損手術 19
X 光攝影 19
微量斷層掃描(Micro-CT Imaging) . 19
樣本收集 20
免疫組織化學染色 20
蘇木素與伊紅染色( hematoxylin-eosin staining, H&E stain) 21
DPPH 自由基清除試驗 . 21
亞鐵離子螯合試驗 21
膠原蛋白檢測 22
重金屬:砷、鉛、鎘、汞、鈣、鎂檢測 . 22
水解胺基酸18 項 23
精胺酸 23
統計分析 24
第参章、實驗結果 25
第肆章、討論 85
第伍章、參考文獻 98

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