游离脂肪酸激活HaCat细胞表面TLR受体并促进创面愈合的初步研究

Preliminary study on the mechanism of active Toll-like receptors on the surface of HaCat cells to promote Tissue-Repair

目的:观察长链游离脂肪酸对体外培养的角质形成细胞的作用,探讨其促进创面愈合的可能机制。方法:①体外培养HaCat细胞,实验分为N、PA、SA、LPS四组,N组为正常对照组,软脂酸(Palmitic Acid,PA)750μmol/L、硬脂酸(Stearic Acid,SA)750μmol/L、脂多糖(lipopolysaccharide,LPS)500ng/L与HaCat细胞共培养72h组分别为PA组、SA组、LPS组;②PA、SA与角质形成细胞共培养72h检测对HaCat细胞NF-κB、MMP-3、MMP-9、VEGF表达的影响。通过免疫印迹法(Weston blot)检测PA、SA、LPS对HaCat细胞TLR2、TLR4、NF-ΚB表达的影响;Real-TimePCR方法检测对HaCat细胞内NF-κB、MMP-3、MMP-9、VEGF表达的影响。结果:SA、LPS激活细胞表面TLR后可以明显促进细胞表面TLR2、TLR4表达及促进细胞内NF-κB、MMP-3、MMP-9、VEGF等细胞因子的表达,从而发挥促进创面愈合的作用。PA促进HaCat细胞内MMP-3、MMP-9、VEGF表达无明显影响(P>0.05)。结论:SA可促进角质形成细胞分泌MMP-3、MMP-9、VEGF等因子促进创面愈合;其机制可能为:SA作用于TLR激活NF-κB信号传导通路促进细胞释放MMP3、MMP-9、VEGF等因素有关。

Objective To investigate the mechanism of free fatty acids （FFA） induced Toll-like receptors activation on the HaCat cell surface to promote Tissue-Repair. Methods Free fatty acids （FFA） and HaCat cell were cultured and divided into N,PA,SA and LPS groups. Group N as control, Palmitic Acid （PA） 750μmol/L, Stearic Acid （SA） 750μmol/L and lipopolysaccharide （LPS）500ng/L were cultured with HaCat cells 72h which were divided into PA,SA and LPS groups. TLR2,TLR4 and NF-κB have been detected by Western blot. MMP-3,MMP-9 and VEGF have been detected by real-time PCR. Results Stearic acid （SA） and LPS can active Toll-like receptors of HaCat cells to promote the expression of TLR-2 and TLR-4 and Tissue-Repair. There is not significant difference in the insulin level of MMP-3, MMP-9,VEGF between control and Palmitic Acid （PA） groups. Conclusion Stearic acid （SA） can active Toll-like receptors on the surface of HaCat cells to promote Tissue-Repair. The mechanism probably is SA activate dendritic and endothelial cells through TLR and result in nuclear translocation of NF-κB and MMP-3,MMP-9 and VEGF secretion