脂多糖诱导人皮肤成纤维细胞表型改变的信号机制

Dermal fibroblasts were obtained from the normal skin of the patients with hypertrophic scar to investigate as follows: After treating the fibroblasts with monoclonal antibody of TLR4 and CD14 receptor seperately and jointly, LPS is used to stimulate them and the changes of the cells' biological characteristics, phenotype index and gene expression profile are observed. In addition, these changes were analysed carefully, comparing with the fibroblasts which were not treated with monoclonal antibody, to investigate the function of TLR4 and CD14 receptor.The activation status of the three ingredients (including p38k, Erk and JNK) of fibroblasts' MAPK signal pathway were detected seperately and the one which made sense would be picked out. We cultured fibroblasts with specific inhibitors of the useful kinase above, added LPS to them, and then observed the changes of the cells' biological characteristics, phenotype index and gene expression profile. Comparison analysis was conducted with the specimen which were not treated by the inhibitor, to confirm the function of these changes in signal transduction.This project was planed to explore the mechanism of changes of human fibroblast's phenotype. The results would create a new way in the early pretention and treatment of hypertrophic scar after burn injury.

采取烧伤患者正常皮肤，行成纤维细胞培养并开展以下研究：①分别及联合使用抗Toll样受体4（TLR4）和CD14受体单克隆抗体处理成纤维细胞后，用细菌脂多糖（LPS）刺激，观察细胞生物学特性、表型指标及基因表达谱的变化。并与未采用单克隆抗体处理标本对比分析，探讨TLR4和CD14受体的地位。②采用LPS刺激成纤维细胞，分别检测细胞内丝裂原活化蛋白激酶（MAPK）信号通路的三个组分p38激酶、细胞外调控激酶（Erk）和c-jun氨基末端激酶（JNK）活化情况，初步筛选出有意义的激酶。③分别采用上述有意义激酶的特异性抑制剂预孵育成纤维细胞，然后再加入LPS，观察细胞生物学特性、表型指标及基因表达谱的变化，并与未采用抑制剂处理标本对比分析，以确认这些激酶变化在信号转导中的意义。本项目从信号通路角度探讨LPS诱导人皮肤成纤维细胞表型改变的机制，研究结果将为烧伤后增生性瘢痕早期防治开辟新的方向。

本研究采用流式细胞仪、透射电镜、免疫组化、免疫荧光、ELISA、RT-PCR、Western Blot等实验技术在体外条件下探索人正常成纤维细胞脂多糖刺激模型下信号通路的改变及相应机制，本研究发现：(1)TLR4和CD14受体参与了脂多糖刺激模型（0.1ug/mL）成纤维细胞的表型改变，成纤维细胞的增殖、分泌TGF-beta1和IFN-gamma、I型III型胶原蛋白的表达、合成及分泌均有TLR4和CD14的重要参与。（2）不同浓度LPS刺激模型发现，随着LPS浓度增高，MAPK/p38呈抑制趋势，而MAPK/JNK和MAPK/Erk均呈激活趋势，此趋势在LPS浓度0.1ug/mL时达到最高点，并在浓度继续增加（0.5ug/mL及1ug/mL）后三个通路均呈抑制趋势，这也与前期研究的最佳刺激浓度一致。（3）分别及联合应用PD98059抑制MAPK/Erk，SP600125抑制MAPK/JNK后发现，MAPK/Erk和MAPK/JNK通路均参与了脂多糖刺激模型下（0.1ug/mL）成纤维细胞的表型改变，成纤维细胞的增殖、分泌TGF-beta1和IFN-gamma、I型III型胶原蛋白的表达、合成及分泌均有MAPK/Erk和MAPK/JNK的重要参与。MAPK信号通路在脂多糖刺激模型下皮肤成纤维细胞表型改变的过程中发挥重要作用。