SPACE肽修饰成纤维细胞生长因子21的制备及对小鼠皮肤瘢痕形成的抑制作用

Preparation of SPACE peptide modified fibroblast growth factor 21 and its inhibitory effect on skin scar formation in mice

目的:制备SPACE肽修饰的成纤维细胞生长因子21(FGF21-SPACE)并探讨其对小鼠皮肤瘢痕形成的抑制作用。方法:将SPACE肽融合表达于FGF21的羧基端,使用大肠杆菌表达系统获得FGF21-SPACE重组蛋白,并制备相应的卡波姆凝胶制剂,聚丙烯酰胺凝胶电泳(SDS-PAGE)与Western blot鉴定其分子量,对重组蛋白携带的组氨酸(His)标签进行免疫组织化学染色,以检测其透皮能力。皮下注射博来霉素诱导小鼠皮肤瘢痕模型,动物实验分为4组,即对照组、模型组、FGF21组、FGF21-SPACE组。HE与Masson染色检测形态学变化;免疫荧光染色检测CD31、TGF-β1表达;Western blot检测α-SMA、纤连蛋白、胶原蛋白Ⅰ的表达。结果:FGF21与FGF21-SPACE的理论分子量分别为25.54 kD与26.61 kD,与SDS-PAGE及Western blot鉴定结果相一致。涂抹FGF21-SPACE凝胶的皮肤组织His染色强度明显强于FGF21凝胶,而空白凝胶则为阴性染色。与FGF21组相比较,FGF21-SPACE组的真皮厚度与纤维化程度降低,CD31表达升高,TGF-β1、α-SMA、纤连蛋白、胶原蛋白Ⅰ表达降低。结论:FGF21-SPACE重组蛋白具备较好的透皮能力,可以抑制皮肤瘢痕形成,促进血管生成,降低纤维化相关因子的表达,具有较好的应用前景。

Objective:SPACE peptide modified fibroblast growth factor 21(FGF21-SPACE)was prepared and its inhibitory effect on skin scar formation in mice was investigated.Methods:The SPACE peptide was fused at the carboxy terminus of FGF21,and the FGF21-SPACE recombinant protein was obtained using an E.coli expression system,and the corresponding carbomer gel was prepared. Sodium dodecyl sulphate polyacrylamide gel electrophoresis(SDS-PAGE)and Western blot(WB)was used to identify its molecular weight. To examine its transdermal ability,immunohistochemical staining was used to detect histidine(His)tag of the recombinant protein. The mouse skin scar model was induced by bleomycin through subcutaneous injection. The animal experiments were divided into 4 groups:control group,model group,FGF21 group and FGF21-SPACE group. HE and Masson staining was used to detect morphological changes;Immunofluorescence staining was used to detect the expression of CD31 and TGF-β1;WB was used to detect the expression of α-SMA,fibronectin and collagen Ⅰ.Results:The theoretical molecular weights of FGF21 and FGF21-SPACE were 25.54 kD and 26.61 kD,respectively,which were consistent with the results of SDS-PAGE and WB. The staining intensity of His in skin tissue applicated with FGF21-SPACE gel was significantly stronger than that of FGF21 gel,while the blank gel was negative staining. Compared with FGF21 group,the thickness of dermis and the degree of fibrosis in the FGF21-SPACE group decreased,the expression of CD31 increased,and the expression of TGF-β1,α-SMA,fibronectin and collagen Ⅰ decreased.Conclusion:FGF21-SPACE recombinant protein has a good transdermal ability,can inhibit skin scar formation,promote angiogenesis,reduce the expression of fibrosis-related factors,and has a good application prospect.