Periostin對機械拉力造成MG-63類骨細胞凋亡反應的 影響

Abstract

適當的機械力刺激有助於骨細胞的增生和分化，過大的力量刺激則會造成細胞的傷害。Periostin為90kDa matricellular proteins的成員，存在於人體含豐富膠原蛋白的結締組織中，包括心臟瓣膜、肌腱、軟骨膜、骨膜和牙周組織等。近期研究已顯示在牙周組織因應機械性力量的變化過程中，Periostin對於組織重塑及維持結構平衡有其重要性，Periostin在細胞凋亡、增生、遷移也扮演了重要角色。 本研究以Adhesion test 及 Wound healing test觀察Periostin在MG-63類骨母細胞貼附及遷移所扮演的角色，並檢測其Periostin蛋白表現在不同程度週期性張力(5%或15%)及TGF-β調控下的反應。實驗方法是以Flexercell® Strain Unit 給予週期性張力，結果發現高強度刺激(15%)會造成細胞發生凋亡。以免疫螢光染色及流氏細胞儀等方式檢測及定量分析凋亡反應，並測量active caspase-3 及 cPARP 活性，以觀測細胞凋亡，並測試Periostin在拉力所引起細胞凋亡反應中所扮演的角色。本研究結果顯示Periostin有助於細胞貼附及遷移;低強度的週期性張力(5%, 0.1Hz)會刺激MG-63細胞Periostin蛋白的表現，而高強度的週期性張力或外加TGF-β抑制劑則會抑制其表現。此外，rhPOSTN會抑制高強度週期性張力所引起細胞凋亡反應及cPARP的表現。本研究證實不同強度的週期性張力對於MG-63類骨母細胞的Periostin蛋白表現具有不同的調控作用， Periostin有助於MG-63細胞貼附及遷移，並對高強度週期性拉力所引起細胞凋亡反應有抑制效果。

Periostin (POSTN), a disulfide-linked 90-kDa matricellular protein, is predominantly expressed in collagen-rich connective tissues such as heart valves, tendons, perichon-drium, periosteum, and periodontal ligament. Previous studies suggested that periostin is essential for connective tissue homeostasis and is important in maintaining the integrity and function of alveolar bone and periodontal ligament in respond to mechanical stress. The role of periostin in cell apoptosis, proliferation, and migration has been speculat-ed. While relevant mechanical loading is pivotal to proliferation and differentiation of osteoblasts, high-level stretch may damage cells or alter cellular responses. In the present study, we investigated the effects of periostin on cell migration and adhesion of MG-63 osteoblastic cells via cell attachment and wound healing test. We further checked the regulation of periostin protein expression with different amplitudes (5% and 15%) of mechanical stretch and TGF-β signaling. The phenomenon of mechanical stretch-induced apoptosis in MG-63 cells was initially assessed with PI, YO-PRO-1, and Hoechst 33342 fluorescence staining, followed by quantitative analysis with flow cy-tometry. Active caspase-3 and cPARP activities were evaluated to monitor a particular route of cell apoptosis. The results revealed that periostin enhanced the ability of migra-tion and adhesion of MG-63 cells. Periostin protein expression was induced by low-level mechanical stretch (5% elongation), while suppressed by high-level mechanical stretch (15% elongation) or TGF-β inhibitor (SB431542). The percentages of apoptotic cells ex-pressing cPARP protein were substantially increased when MG-63 cells were subjected to 15% cyclic mechanical stretch for 48 hrs. Moreover, the mechanical stretch-induced apoptosis was inhibited in the presence of exogenous rhPOSTN. In conclusion, our pre-sent study suggests that periostin could play a protective role thin mechanical stretch-induced apoptosis of osteoblastic cells.