循環拉伸對大鼠脂肪幹細胞分化成軟骨細胞的影響

退化性關節炎是常見的骨關節疾病，關節軟骨無血管的結構導致其自我修復不易，現今組織工程用於關節軟骨修復為最具潛力的治療方式。文獻報導循環拉伸應力刺激具有調節細胞增殖及分化的能力。本研究旨在探討TGF-β1誘導大鼠脂肪幹細胞軟骨分化環境，循環拉伸刺激對軟骨分化的影響。本研究以群落形成能力確認大鼠脂肪幹細胞增殖能力，及鑑定軟骨和硬骨分化能力後進行實驗。將初代大鼠脂肪幹細胞貼附於具有第一型膠原蛋白修飾的矽膠薄膜表面，以拉伸應變10 %，及拉伸頻率為1.0 Hz或0.5 Hz的循環拉伸，每天連續刺激3小時(為期3天)，並與靜態培養且無分化控制組及靜態培養且分化組比較。實驗結果發現，頻率1.0 Hz循環拉伸刺激下細胞產生大量的凋亡小體。頻率0.5 Hz循環拉伸刺激則可產生直徑100-150 μm細胞團塊，由Safranin O染色證實代表軟骨分化的酸性蛋白多醣存在，醣胺聚醣(GAGs)濃量相對於靜態培養組較高且具有統計差異。透過蛋白質分析腫瘤抑制蛋白p53、cleaved caspase-9、cleaved caspase-3及phospho-NF-κB p65在細胞的表現，結果顯示，1.0 Hz的拉伸頻率會造成內源性細胞凋亡途徑的活化，使caspase-9及caspase-3被裂解導致細胞死亡，且p53參與在其中，而頻率0.5 Hz循環拉伸刺激僅活化NF-κB細胞生存途徑使p65轉錄因子磷酸化。透過反轉錄聚合酶連鎖反應分析基因，比較靜態分化組與頻率0.5 Hz循環拉伸刺激分化組Sox-9及RUNX2的表現，發現循環拉伸刺激下調節軟骨分化Sox-9基因表現較高，而會造成細胞肥大化的RUNX2則有較低的表現。雖然本研究尚未完全釐清循環拉伸刺激促進大鼠脂肪幹細胞軟骨分化的訊息傳遞途徑，但由實驗結果顯示以應變10 % 每天連續拉伸3小時條件下(為期3天)，頻率0.5 Hz循環拉伸刺激具有促進軟骨分化的效果，而頻率1.0 Hz則會活化內源性細胞凋亡途徑造成細胞死亡且腫瘤蛋白p53參與在其中。

關鍵字

骨關節炎；大鼠脂肪幹細胞；軟骨分化；循環拉伸；腫瘤抑制蛋白p53

並列摘要

Osteoarthritis is the most common form of arthritis, affecting millions of people worldwide. Articular cartilage is an avascular tissue with limited ability to repair and self-renewal. Cartilage tissue engineering is a promising modality for repairing damaged and defective cartilage tissue today. It has been reported that cyclic stretching stress stimulates the proliferation and differentiation of stem cells. The goal of this study was to investigate the effects of cyclic stretching on TGF-β1-induced chondrogenic differentiation of rat adipose-derived stem cells (rADSCs). In the present study, The proliferation ability of rADSCs was confirmed by colony-forming unit assay, and the chondrogenic and osteogenic differentiation were also confirmed. For cyclic stretching, 10 % of strain and two frequencies of 1.0 Hz or 0.5 Hz were applied on a type Ⅰ collagen-grafted silicone membrane in which rADSCs were adhered. The stretching was applied for 3 hours per day for a period of 3 days, and compared with two static culture groups with or without differentiation induction. At stretching frequency of 1.0 Hz, the majority of the cells showed apoptotic bodies, while the stretching frequency of 0.5 Hz resulted in cell aggregates having a diameter of 100-150 μm, and acidic proteoglycans stained by Safranin O was detected representing chondrogenic differentiation. The concentrations of glycosaminoglycans (GAGs) in the culture mediaa was significantly higher than two static cultured groups. The protein expression level of tumor suppressor protein p53, cleaved caspase-9, cleaved caspase-3 and phospho-NF-κB p65 in rADSCs were revealed by western blot analysis. It was found that, when the stretching frequency of 1.0 Hz was applied to rADSCs, the intrinsic apoptosis signaling pathway along with the involvement of tumor suppressor protein p53 was activated, and cleavage caspase-9 followed by cleavage of procaspases-3 caused the cell death. However, the stretching frequency of 0.5 Hz only activated the NF-κB survival signaling pathway, e.g. the phosphorylation of transcription factor p65. In the reverse transcription-polymerase chain reaction (RT-PCR), it was found that, the stretching frequency of 0.5 Hz gave higher gene expression of cartilage differentiation gene Sox-9 and lower gene expression of cellular hypertrophy gene RUNX2, compared with the differentiation static culture group. Although the signal transduction pathway of cyclic stretching to promote the chondrogenic differentiation of rADSCs is not fully discovered in the present study, the results indicate that under the cyclic stretching strain of 10 % for 3 hours per day for a period of 3 days, the stretching frequency of 0.5 Hz gave rise to promoting chondrogenic differentiation of rADSCs and that of 1.0 Hz activated the intrinsic apoptosis signaling pathway along with tumor suppressor protein p53 causing cell death.

並列關鍵字

Osteoarthritis ； rat adipose-derived stem cells ； chondrogenic differentiation ； cyclic stretching ； tumor suppressor protein p53

參考文獻

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