探討結締組織生長因子CTGF刺激單核球細胞所需之細胞表面分子

Investigation of cell surface molecules essential for CTGF stimulation on monocytes

邵彥豪(Yen-Hao Shaw)

指導教授 ： 陳玫潔

臺北醫學大學/醫學院/醫學科學研究所/碩士(2010年)

摘要

結締組織生長因子，屬於分泌型蛋白CCN家族的一員，在細胞及細胞外基質交互作用中扮演重要的角色，其主要功能在於調控細胞分化、增生、黏附、移動、凋亡、纖維化、血管新生及胞外基質的生成等；在本實驗室先前的研究中已知，結締組織生長因子會引發人類CD14+單核球細胞提升IL-8的分泌。然而，結締組織生長因子如何從胞外傳遞訊息至胞內活化訊息傳遞路徑的過程目前尚不清楚，是否經由細胞表面的醣類或特殊受體亦未可知。在此我們檢視細胞表面的醣類fibronectin、heparin和heparan sulfate與CTGF的交互作用，發現Heparin sulfate proteoglycans參與CTGF之刺激作用。為了進一步鑑定其受體，首先以integrin ??2次單位的拮抗性抗體來觀察細胞膜上的integrin是否為CTGF的特殊受體；而後使用TrkA磷酸化的抑制劑K-252a，檢視TrkA是否參與CTGF引發IL-8分泌過程；最後利用乳鐵蛋白與CTGF競爭LRP1結合位置以辨別LRP1之重要性。為了解CTGF是否經由胞吞作用來傳遞訊息，使用胞吞作用的抑制劑Phenylarsine Oxide及核內體抑制劑NH4Cl以觀察其對CTGF刺激細胞產生IL-8之影響。結果顯示在人類CD14+單核球細胞中，CTGF可能藉由結合LRP1及TrkA，並利用胞吞作用進入細胞，經過核內體的消化代謝後，接著活化p38、ERK及JNK的MAPK訊息傳導路徑，最終刺激IL-8的分泌提升。

關鍵字

結締組織生長因子 ； 單核球細胞

並列摘要

Connective Tissue Growth Factor (CTGF), is one member of secretory protein CCN family that mediates the interaction of cell surface and extracellular matrix (ECM). Among the many function of CTGF are angiogenesis, adhesion, osteogenesis, tissue repair, fibrosis, proliferation, migration, and differentiation, CTGF is also a pathogenic factor in variety of fibrotic disorders. In our previous study, CTGF has been found to induce IL-8 secretion on human CD14+ monocytes. However, the signal transduction pathway of CTGF and the specific receptor recognized by CTGF on human monocytes are still poorly understood. In this study, we investigated the interaction between surface fibronectin, heparin, heparan sulfate and CTGF, found heparin sulfate proteoglycans involved in CTGF function. To further identify the receptor of CTGF, we used antagonist of integrin ??2 to define whether integrin ??2 is the specific receptor for CTGF. Then we utilized TrkA inhibitor K-252a to identify whether TrkA participate in the IL-8 secretion pathway induced by CTGF. Finally we used lactoferrin competed with CTGF binding to LRP1 to clarifying whether LRP1 is the receptor for CTGF. Furthermore, we utilized additional endocytosis inhibitor Phenylarsine Oxide and endosome inhibitor NH4Cl to identify whether IL-8 induction through endocytosis. Data suggested that on human CD14+ monocytes, CTGF might bind to LRP1 and TrkA then internalized into cells by endocytosis, combined with endosome then activated MAP Kinase p38, ERK and JNK eventually increased IL-8 secretion.

並列關鍵字

CTGF ； monocytes

參考文獻

1. Brigstock, D.R., et al., Proposal for a unified CCN nomenclature. Mol Pathol, 2003. 56(2): p. 127-8.

2. Lau, L.F. and S.C. Lam, The CCN family of angiogenic regulators: the integrin connection. Exp Cell Res, 1999. 248(1): p. 44-57.

3. Brigstock, D.R., The CCN family: a new stimulus package. J Endocrinol, 2003. 178(2): p. 169-75.

4. Chen, C.C., N. Chen, and L.F. Lau, The angiogenic factors Cyr61 and connective tissue growth factor induce adhesive signaling in primary human skin fibroblasts. J Biol Chem, 2001. 276(13): p. 10443-52.

5. Bork, P., The modular architecture of a new family of growth regulators related to connective tissue growth factor. FEBS Lett, 1993. 327(2): p. 125-30.