Analysis of the stimulatory effect of sphingolipid, especially SPC, on the wound healing

鞘脂尤其是SPC对伤口愈合的促进作用分析

• 批准号: 11670850
• 负责人: TSUBOI Ryoji
• 金额: $ 0.9万
• 依托单位: JUNTENDO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670850/
• 关键词: SPC; wound healing; sphingo lipid; wound contraction; extracellular matrix; 細胞外マトリックス; フフィンゴ脂質

Sphingosylphosphorylcholine (SPC) is a sphingolipid metabolite known to stimulate healing by virtue of its potent mitogenic effect on various cells. To clarify the mechanism of SPC on the healing process, we examined the effect of SPC on wound contraction using an in vitro model and on the synthesis of fibronectin (FN), an extracellular matrix protein, in human dermal fibroblasts.(1) A mixture of human dermal fibroblasts and porcine type I collagen in a serum-free medium was gelled. The effect of SPC was analyzed by measuring the amount of contracted surface area.SPC stimulated collagen gel contraction dose dependently, maximal effect at 10 uM of SPC.The effect of SPC was not 'Inhibited by pre-treatment with anti-TGF-b or anti-PDGF-BB antibodies.Among the various signal transduction inhibitors, pertussis toxin, staurosporine and H7 were found to inhibit the action of SPC whereas genistein and tyrphostin A47 were not, suggesting that fibroblast contraction induced by SPC is mediated by a trimeric GTP-binding protein (G protein)-coupled receptor and protein kinase.(2) SPC was found dose-dependently to up-regulate the expression of protein and mRNA levels of FN.The effect of SPC on the expression of TGF-b, PDGF-BB, IL-4 and IL-6 in fibroblasts was then examined. Among the tested cytokines, the mRNA expression and protein level of only IL-6 was significantly up-regulated by the application of SPC in a dose dependent manner. Furthermore, northern blotting analysis revealed that the mRNA level of FN in fibroblasts was up-regulated by the addition of recombinant IL-6 and suppressed by the addition of neutralizing antibody against IL-6, suggesting that SPC promoted the synthesis of FN partially through IL-6 action.These results suggest that stimulatory effect of SPC on wound healing is exerted by the stimulation of wound contraction and extracellular matrix synthesis as well as the stimulation of reepithelialization.

鞘鞘酰基磷酰胆碱（SPC）是一种鞘脂代谢物，已知通过其对各种细胞的有丝分裂作用来刺激愈合。为了阐明SPC在愈合过程中的作用机制，我们利用体外模型研究了SPC对伤口收缩的影响，以及SPC对人真皮成纤维细胞中纤维连接蛋白（一种细胞外基质蛋白）合成的影响。(1)将人真皮成纤维细胞与猪I型胶原的混合物在无血清培养基中凝胶化。通过测量收缩表面积的大小来分析SPC的效果。SPC刺激胶原凝胶收缩呈剂量依赖性，在10 μ m时效果最大。抗tgf -b或抗pdgf - bb抗体预处理不抑制SPC的作用。在多种信号转导抑制剂中，百日毒、staurosporine和H7对SPC有抑制作用，染料木素和tyrphostin A47对SPC无抑制作用，提示SPC诱导成纤维细胞收缩是由gtp结合蛋白（G蛋白）三聚体偶联受体和蛋白激酶介导的。(2) SPC可呈剂量依赖性上调FN蛋白和mRNA的表达水平。检测SPC对成纤维细胞TGF-b、PDGF-BB、IL-4、IL-6表达的影响。在所检测的细胞因子中，只有IL-6的mRNA表达和蛋白水平被SPC显著上调，且呈剂量依赖性。此外，northern blotting分析显示，加入重组IL-6可上调成纤维细胞中FN的mRNA水平，而加入针对IL-6的中和抗体可抑制FN的mRNA水平，表明SPC通过IL-6的作用部分促进了FN的合成。这些结果表明，SPC对创面愈合的刺激作用是通过刺激创面收缩和细胞外基质合成以及刺激再上皮化来实现的。

项目成果

Suhr KB,Tsuboi R,Ogawa H: "Sphingosylphoshorylcholine stimulates contraction of fibroblast-embedded collagen gel"Br J Dermatol. 143. 66-71 (2000)

Suhr KB、Tsuboi R、Okawa H：“鞘氨醇磷酰胆碱刺激成纤维细胞嵌入的胶原凝胶的收缩”Br J Dermatol。

Snhr KB,Tsuboi R.Ogawa H: "Sphingosylphoshorylcholine stimulates contraction of fibroblast-embedded collagen gel"Br J Dermatol. 143. 66-71 (2000)

Snhr KB、Tsuboi R.Okawa H：“鞘氨醇磷酰胆碱刺激成纤维细胞嵌入的胶原凝胶的收缩”Br J Dermatol。

Suhr KB, Tsuboi R, Ogawa H: "Sphingosylphoshorylcholine stimulates contraction of fibroblast-embedded collagen gel."Br J Dermatol. 143. 66-71 (2000)

Suhr KB、Tsuboi R、Okawa H：“鞘氨醇磷酰胆碱刺激成纤维细胞嵌入的胶原凝胶的收缩。”Br J Dermatol。