探討軟骨硫素誘導 ATDC5 軟骨前驅細胞分化之分子機轉

軟骨組織之細胞外間質 (extracellular matrix, ECM) 中含有大量之黏多醣 (glycosaminoglycan, GAG)，具有保水、維持關節的柔軟與強韌性之功能，然而現今報導黏多醣具有影響或協同調節細胞生長及分化之作用。依照黏多醣之組成雙醣單元不同而有不同之型態，包括肝硫素 (heparan sulfate, HS)、軟骨硫素 (chondroitin sulfate, CS) 等，其影響細胞功能也有所不同。在此我們確認了軟骨硫素及肝硫素可促進 ATDC5 軟骨前驅細胞 (chondroprogenitor cell) 之貼附、生長以及分化成軟骨細胞 (chondrogenesis)。其中軟骨硫素之ㄧ chondroitin-6-sulfate (C-6-S) 促進軟骨細胞分化之能力比過往使用之誘導物－胰島素 (insulin) 還佳。此外，改變最初細胞培養密度會影響軟骨硫素誘導軟骨細胞分化之時程，在高密度細胞培養模式會加速軟骨細胞分化成熟為肥大軟骨細胞 (hypertrophic chondrocyte) 而喪失原有軟骨細胞特性；相反的，以低密度細胞培養可維持軟骨細胞特性長達 23 天。有趣的是，在軟骨硫素誘導分化過程中發現細胞並無聚集現象 (condensation)。但其不影響參與細胞聚集分子－ Sox9、N-cadherin 基因之表現。因此，軟骨硫素具有模擬細胞聚集進而誘導軟骨細胞分化之作用。最後，我們進一步探討 C-6-S 誘導 ATDC5 細胞分化之細胞內訊號傳遞因子，首先證實 C-6-S 透過 mitogen-activated protein kinase (MAPK) 中 p38 訊號傳遞誘導 ATDC5 軟骨細胞分化。本篇研究證實軟骨硫素具有促進細胞貼附、生長以及誘導軟骨細胞分化的功能。因此，軟骨硫素未來可應用於軟骨組織工程來達到臨床之軟骨組織修復與再生。

關鍵字

黏多醣；細胞外間質；軟骨細胞分化；軟骨硫素；肝硫素

並列摘要

The cartilage extracellular matrix (ECM) contains large amount of glycosaminoglycan (GAG), which has water-holding property, contributes resilience and resistance to joint deformation. The most common GAG including chondroitin sulfate (CS), keratan sulfate (KS), and heparan sulfate (HS) has a unique structure consisting of repeating disaccharide units. Many recent reviews indicated that GAG mediates cell activity, such as proliferation, differentiation and migration, implicating critical roles of GAG play in critical biological processes. Our current experiments showed that CS and HS could mediate ATDC5 chondroprogenitor cell adhesion, proliferation, and chondrogenesis. 6-O-sulfated CS (chondroitin-6-sulfate, C-6-S) promotes chondrogenesis more efficiently than insulin, which was used as an inducer for ATDC5 chondrogenic differentiation. In addition, initial cell density can affect C-6-S-induced chondrogenesis. High inoculation cell density could accelerate chondrogenic differentiation and promote maturation into hypertrophic chondrocyte. In contrast, low density culture could maintain chondrocytic characteristics for a long term culture of 23 days. More interesting, chondrogenesis of ATDC5 cells induced by C-6-S skipped the cellular condensation phenotypes. But it did not affect the expressions of condensation related molecules, Sox9 and N-cadherin. Moreover, the intracellular signal pathways mediating C-6-S induced chondrogenesis was also studies. It indicated that C-6-S induced chondrogenesis via p38 signal pathway. Our studies demonstrated the induction role of C-6-S in chondrogenesis of ATDC5 cells. The obtained information will be helpful on the application of using C-6-S for cartilage repair and for stem cell therapy in cartilage tissue regeneration.

並列關鍵字

chondroitin sulfate ； heparan sulfate ； extracellular matrix ； glycosaminoglycan ； GAG ； CS ； chondrogenesis ； p38 ； MAPK

參考文獻

1. Lefebvre, V. and P. Smits, Transcriptional control of chondrocyte fate and differentiation. Birth Defects Res C Embryo Today, 2005. 75(3): p. 200-12.

2. Adams, C.S. and I.M. Shapiro, The fate of the terminally differentiated chondrocyte: evidence for microenvironmental regulation of chondrocyte apoptosis. Crit Rev Oral Biol Med, 2002. 13(6): p. 465-73.

3. Goldring, M.B., K. Tsuchimochi, and K. Ijiri, The control of chondrogenesis. J Cell Biochem, 2006. 97(1): p. 33-44.

4. Shukunami, C., C. Shigeno, T. Atsumi, K. Ishizeki, F. Suzuki, and Y. Hiraki, Chondrogenic differentiation of clonal mouse embryonic cell line ATDC5 in vitro: differentiation-dependent gene expression of parathyroid hormone (PTH)/PTH-related peptide receptor. J Cell Biol, 1996. 133(2): p. 457-68.

5. Huang, W., U.I. Chung, H.M. Kronenberg, and B. de Crombrugghe, The chondrogenic transcription factor Sox9 is a target of signaling by the parathyroid hormone-related peptide in the growth plate of endochondral bones. Proc Natl Acad Sci U S A, 2001. 98(1): p. 160-5.

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探討軟骨硫素誘導 ATDC5 軟骨前驅細胞分化之分子機轉

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