藉由週期性張力之刺激於人類MG63類成骨母細胞及正常骨衍生細胞 引發缺氧可誘導因子-1α及血管內皮生長因子的產生

Abstract

引發缺氧可誘導因子HIF-1α及血管內皮生長因子VEGF的產生，主要與血管新生的機制有關，此機制參與了如修復組織缺氧、骨骼重塑，甚或腫瘤生成的過程，其中骨骼重塑的複雜機轉則長期為引人關注的研究方向之一。目前已知：機械力量刺激在骨骼重塑的機制上扮演著重要的調節角色，且藉由機械力量刺激也被證實可於人類牙周韌帶纖維母細胞上引發血管新生的過程，然而利用機械力量刺激於成骨母細胞上，是否亦會引起血管新生的機制，並進一步影響骨骼重塑的路徑目前則有待證實。因此本研究首先利用MG63類成骨母細胞株作為實驗對象，施予週期性張力刺激三天後觀察細胞表徵之變化，並以半定量反轉錄-聚合酶鏈鎖反應分析COX-2、HIF-1α及VEGF的表現，結果發現：三者的mRNA level在機械張力刺激下的先後表現為COX-2最先（60分鐘後），HIF-1α其次（24-48小時），而VEGF可能同時受兩者影響，分別於5小時及48小時有兩次上升表現。再以西方點墨法分析其蛋白質的表現量，結果三者上升表現之順序為COX-2最先（5小時），HIF-1α其次（5-48）小時，而VEGF最後（48小時）。另外再以同樣之張力條件，刺激於正常成骨母細胞上24小時並觀察其變化，結果發現其基因與蛋白質之上升表現皆早於MG63細胞，在mRNA level為 COX-2：10分鐘，VEGF：10分鐘，HIF-1α：5小時，而蛋白質表現為COX-2：10分鐘，HIF-1α：5小時，VEGF：10分鐘與24小時。由本實驗結果證實：藉由張力刺激於兩種成骨母細胞上可誘導HIF-1α及VEGF的產生，且成骨母細胞分化程度之不同亦會影響其對張力刺激的反應，由此可推論經由機械張力刺激所引發之血管新生與骨骼重塑機轉應廣泛地存在於人類成骨母細胞中。

Induction of hypoxia inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) is mainly involved in angiogenesis, which is required for many biological and/or pathological procedures, such as repairing ischemic tissue, bone remodeling, and cancer metastasis. Among them, bone remodeling is the most relevant issue in orthodontics. It is widely accepted that mechanical stimuli play an important role in bone remodeling during growth and development. Also, it had been proved that mechanical stress would induce production of angiogenic regulators in cultured human periodontal ligament fibroblasts. However, whether mechanical stress would have similar effects on human osteoblastic cells remains to be addressed. In this study, two different populations of osteoblast-like cells were used: human osteosarcoma cell line-MG63 and human bone derived primary cells, to receive mechanical stimuli. Morphological changes of these cells after mechanical stress was recorded. Expression of Cyclooxygenase-2 (COX-2), HIF-1α and VEGF was analyzed by semi-quantitative PT-PCR and western blotting. Our results showed that in MG63 cells with mechanical stretching, COX-2 and HIF-1α could be induced sequentially at 1 hour and one day respectively, and then followed by VEGF with biphasic increase at 5 hour and 2 days. The production of VEGF seems to relate to increase of COX-2 since COX-2 inhibitor could decrease the expression of VEGF significantly. These findings were similar in the bone derived cells thought with a quicker response to mechanical stress than MG63 cells. Therefore, the results indicate that mechanical stress could induce the production of HIF-1α and VEGF in human osteoblasts and the responses to mechanical stress might depend on the differentiation status of cells.