利用快速篩選平台分析鍶化合物對骨細胞的影響

Abstract

硫酸鈣是石膏的主要成分，可於外科醫學上作為患部固定、支撐的用途，也是重要的骨替代材料，具備生物相容性和良好的骨傳導性，在移植後不會引起發炎反應，幫助患部進行骨質新生。而在先前的研究中指出，添加鍶離子具有抗骨質疏鬆的效果，能刺激成骨前驅細胞分化成熟成成骨細胞以促進骨質新生，同時抑制蝕骨細胞進行骨質再吸收，以達到抑制骨質流失的效果，因此研究鍶離子在不同條件下對於誘導成骨細胞分化的能力就顯得相當重要，然而以往的生物測試無法同時快速、方便地檢驗鍶離子在不同條件下誘導成骨前驅細胞的效率，因此本研究的目的是要建立以冷光系統作為快速篩檢的平台，檢測並量化特定環境對於成骨細胞分化的影響。首先在成骨前驅細胞株MC3T3-E1培養時加入骨塑型蛋白和成骨誘導培養基進行成骨誘導，以西方墨點法確定成骨前驅細胞的分化上游調控蛋白質RUNX2表現量有顯著提高。然後以鹼性磷酸酶染色觀察到受到誘導的細胞有明顯變色，即開始進行分化作用，同時以成骨細胞分化的重要指標骨鈣蛋白的啟動子作為冷光報導載體的主架構，轉染進細胞並誘導分化，觀察到其在受到誘導的環境下能表現出較高的冷光讀值，確立此冷光系統具有檢測待測環境是否具有骨誘導作用的能力，並可作為鑑定該環境是否有助於成骨細胞分化的指標。接著將細胞培養在不同鍶離子濃度的環境下，可觀察到其因受到不同程度的誘導而表現冷光。在上述系統能穩定表現後，為求能在眾多不同的環境下進行測試，將上述冷光系統以慢病毒感染MC3T3-E1細胞株製作穩定細胞株，並測試此穩定細胞株是否能符合先前的誘導實驗結果，以便將來能應用在快速篩檢大量不同的骨替代材。

Calcium sulfate is commonly used as bone graft that can fix and support bone fracture in surgical medication. Its biocompatibility and osteoconductivity allows injury-site to recover without causing inflammatory reactions after transplantation. In previous studies, the addition of strontium ions can relieve osteoporosis by stimulating maturation of preosteoblasts via RUNX2 activation, a key transcription activator of osteoblast differentiation, to promote bone regeneration. RUNX2, as an upstream regulatory protein of bone cell maturation, can upregulate the expression of downstream proteins such as osteopontin and osteocalcin, which represent the beginning of calcification among mature osteoblasts. On the other hand, strontium ions can also inhibit bone resorption through inactivating the functionality of osteoclasts. Therefore, studying the dual effect of strontium ions in medical components or bone grafts becomes important nowadays. However, it is too difficult to screen effects of strontium components or biomaterials fast, easily and quantifiably in cell culturing experiments under numerous conditions. The purpose of this study is to establish a quick screening platform through luciferase reporter system that could distinguish effects of different components and materials to preosteoblasts at the same time. First, preosteoblast MC3T3-E1 cells were induced with bone morphogenetic proteins 2 and osteogenic induction medium for bone formation, and the expression of RUNX2 was examined using immunoblotting. Moreover, the osteocalcin promoter was fused to luciferase gene to generate the reporter plasmid pGL3-OC1316 and the promoter activity can be enhanced by osteoinduction. A series deletion analysis of the reporter plasmids showed that the reporter plasmid pGL3-OC343 containing six copies of RUNX2-binding sites with strong enhancement by osteoinduction. In addition, the luciferase activities of the osteocalcin reporter plasmids were enhanced by specific concentration of strontium ions under osteoinduction. Finally, this study will use lentivirus infection to construct stable cell line by using osteocalcin reporter plasmids in MC3T3-E1 cells, which will be used to quick screen the effects of different bone grafts or biomaterials.