转化生长因子-β1噬菌体模拟肽促进成纤维细胞增殖的效果

Proliferative effect of TGF-β1 phage model peptide on fibroblasts

目的从噬菌体随机12肽库中筛选获得转化生长因子β1(TGF-β1)噬菌体模拟肽,评估其促进人正常皮肤成纤维细胞增殖的效果。方法以人TGF-β1单克隆抗体为靶,生物淘选噬菌体模拟肽。选择其中一组与TGF-β1基因序列相似度最高的噬菌体模拟肽,设定4组,阴性对照组、噬菌体M13对照组、TGF-β1对照组和噬菌体模拟肽组。噻唑蓝(MTT)比色法定量测定活细胞数量以检测细胞的增殖。免疫荧光方法检测噬菌体模拟肽与成纤维细胞的亲和力。实时定量PCR分析方法检测成纤维细胞I型胶原蛋白(COL1)、Ⅲ型胶原蛋白(COL3)和结缔组织生长因子(CTGF)的mRNA表达水平。结果共获得10种噬菌体模拟肽,选择与TGF-β1基因序列相似度最高的第一组噬菌体模拟肽进行实验。MTT结果显示,噬菌体模拟肽组能够促进成纤维细胞增殖(P<0.05)。免疫荧光结果显示,噬菌体上的模拟肽,而非噬菌体本身,能够与成纤维细胞结合;实时定量PCR的结果显示,48 h噬菌体模拟肽组COL1和COL3 mRNA的表达水平较阴性对照组和噬菌体M13对照组均明显增高(P<0.05),而CTGF的mRNA表达水平较阴性对照组和噬菌体M13对照组2 d时明显增高(P<0.05),3 d时表达有所降低(P<0.05)。结论噬菌体模拟肽可能是通过调节CTGF的表达,调节成纤维细胞的增殖。

Objective To isolate transforming growth factor-beta1（ TGF-β1） phage model peptides from phage display12-mer peptide library,and to evaluate their proliferative effect on normal skin fibroblasts. Methods Phage 12-mer display peptide library was screened with monoclonal anti-human TGF-β1 to get specific phage model peptides. The model with sequence most similar to TGF-β1 was chosen and 4 groups were enrolled： negative control group,MP13 control group,TGF-β1 control group,and phage model peptide group. MTT assay was used for the quantitative determination of cellular proliferation. Immunofluorescence assay was employed to showthe affinity of the model peptide on skin fibroblasts. Quantitative Real-time PCR analysis was carried out to detect the expressions of I collagen（ COL1）,III collagen（ COL3） and connective tissue growth factor（ CTGF）. Results A total of 10 phage model peptides were obtained. The first one with sequence most similar to TGF-β1 was chosen. The MTT results showed that the phage model peptide could promote fibroblast proliferation（ P〈0. 05）. Immunofluorescence assay revealed that the model peptide on phages rather than phages could bind to the fibroblasts（ P〈0. 05）. The Real-time PCR analysis suggested that the relative expressions of COL1 and COL3 significantly increased in the model group than in the negative control group and M13 group（ P〈0. 05）. The expression of CTGF mRNA increased dramatically after treatment withphage model peptide,and reached the peak at 2 d（ P〈0. 05）. Conclusion The phage model peptide isolated from phage 12-mer display peptide library can promote the proliferation of normal skin fibroblasts by regulating the expression of CTGF.