重组融合饰胶蛋白拮抗转化生长因子β_1刺激增生性瘢痕成纤维细胞增殖的作用

Antagonistic effect of recombinant fusion protein decorin on TGF β_1 stimulating hypertrophic scar fibroblasts

目的 观察重组谷胱甘肽S -转移酶与饰胶蛋白的融合蛋白 (GST -Decorin)对转化生长因子 β1 刺激增生性瘢痕成纤维细胞的作用 ,探索瘢痕治疗的新方法。 方法 采用RT -PCR技术克隆饰胶蛋白 (Decorin)cDNA并连接到pGEX - 4T - 1载体上 ,大肠杆菌内表达GST -Decorin ;体外分离培养增生性瘢痕成纤维细胞 ,加入 1∶6 .2 5 ,1∶12 .5 0 ,1∶2 5 .0 0 ,1∶5 0 .0 0 ,1∶10 0 .0 0稀释度的GST -Decorin ,经四唑盐 (MTT)比色法测定细胞增殖速度 ;进一步将 2mg L的TGF - β1 或同时与 1∶12 .5 0的GST -Decorin加入培养基内 ,比较细胞增殖速度的变化 ,并应用原位杂交及图像分析观察细胞Ⅰ、Ⅲ型前胶原mRNA的表达。 结果 稀释度为 1∶6 .2 5～ 1∶2 5的GST -Decorin可有效地抑制瘢痕成纤维细胞的增殖速度 (P <0 .0 1或P <0 .0 5 ) ;1∶12 .5 0的GST -Decorin能完全拮抗 2mg L的TGF - β1 刺激细胞增殖及Ⅰ、Ⅲ型前胶原的mRNA表达。 结论 GST -Decorin可抑制体外培养的增生性瘢痕成纤维细胞增殖并拮抗TGF - β1 。

Objective To investigate the effect of recombinant glutathione S transferase (GST) Decorin on TGF β 1 stimulating hypertrophic scar fibroblasts. Methods Decorin cDNA was cloned by RT PCR and inserted to pGEX 4T 1 that was then transfected into escherichia coli to express GST Decorin fusion protein. Fibroblasts divided from hypertropnic scar tissue were cultured with serial dilutions of GST Decorin and the cells proliferation rates were measured by MTT colorimetric determination. Furthermore, the changes of cell proliferation rates and mRNA expressions of type Ⅰ and Ⅲ procollagens shown by in situ hybridization and image analysis were observed after GST-Decorin of 1:12.50 dilution and TGF β 1 of 2 mg/L was added together to the culture media. Results The proliferation rates of fibroblasts were significantly inhibited by addition of GST-Decorin to the culture media at 1∶6.25 to 1∶25.00 dilutions ( P <0.01 or P <0.05 vs control groups without GST Decorin). The increases of cell proliferation rates and mRNA expressions of type Ⅰ and Ⅲ pro collagens stimulated with TGF β 1 of 2 mg/L were antagonized completely with GST Decorin of 1∶12.50 dilution. Conclusions Recombinant GST Decorin can inhibit the proliferation of hypertrophic scar fibroblasts and antagonize the effect of TGF β 1 in vitro, indicating that the recombinant fusion protein may be effective in treatment of hypertrophic scar.