摘要
目的从噬菌体随机12肽库中筛选获得转化生长因子（TGF）β1噬菌体模拟肽，评估其抑制瘢痕疙瘩成纤维细胞增殖的作用。方法以人TGF—β1单克隆抗体为靶，生物淘选噬菌体模拟肽。噻唑蓝（MTT）比色法定量测定活细胞的数量。Annexin V—FITC／PI凋亡检测试剂盒和流式细胞仪检测对成纤维细胞的凋亡作用。免疫荧光测定检测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核因子κB（NF—κB），结缔组织生长因子（CTGF）的表达水平。结果共获得10种噬菌体模拟肽，具有与TGF—β1、TGF—β2、TGF—β受体Ⅱ（TβRⅡ）、TGF—β诱导因子、NF—κB或细胞分裂原素活化蛋白激酶（MAPK）相似的序列。MTT比色测定结果显示4种（第7～10组）噬菌体模拟肽组能够抑制瘢痕疙瘩成纤维细胞增殖（P〈0．05）。免疫荧光测定显示是噬菌体上的模拟肽，而不是噬菌体本身，能够与瘢痕疙瘩成纤维细胞相结合；凋亡实验显示这4种噬菌体模拟肽能够轻度促使瘢痕疙瘩成纤维细胞发生轻度的晚期凋亡；实时定量PCR的结果显示这4种噬菌体模拟肽NF—κB的表达降低，表达量分别是阴性对照组的0．28、0．26、0．46、0．30倍，4种噬菌体模拟肽CTGF的表达降低，表达量分别是阴性对照组的0．26、0．60、0．34、0．17倍。结论噬菌体模拟肽可能是通过调节NF—κB及CTGF的表达，调节瘢痕疙瘩成纤维细胞的增殖。
Objective To isolate the transforming growth factor-beta 1 （TGF-β1） phage model peptides from phage 12-mer display peptide library to inhibit the proliferation of keloid fibroblasts. Methods The phage display 12-mer peptide library was screened for 4 rounds with monoclonal anti-human TGF-β1 as the target to yield the specific phage model peptides. The 3-（ 4, 5-dimethyhhiazol-2-yl ）-2, 5- diphenyltetrazolium bromide （MTT） assay was used for the quantitative determination of cellular proliferation. Apoptosis was detected by the Annexin V-FITC/PI apoptosis detection kit and the cells were analyzed with flow cytometry. Immunofluorescent assay was employed to show the binding affinity of model peptides for keloid fibroblasts. Quantitative real-time polymerase chain reaction （PCR） was performed to detect the expressions of nuclear factor kappa B （NF-κB） and connective tissue growth factor （CTGF）. Results Ten phage model peptides were obtained and they were similar to TGF-β1, TGF-[32, TGF-β receptor Ⅱ （TβRⅡ）, TGF-β-induced factor, NF-κB or mitogen-activated protein kinase （MAPK）. The resuhs of MTY showed that four phage model peptides （No. 7 - 10） could inhibit the proliferation of keloid fibroblasts （P 〈 0. 05 ）. The results of apoptotic assessment showed that phage model peptides （No. 7 - 10） could slightly trigger the late apoptotic stage of keloid fibroblasts. The data of immunofluorescence assay revealed that the model peptides on phages rather than phages could bind to keloid fibroblasts. The findingsof quantitative real-time PCR analysis suggested that the relative expression of NF-κB decreased in phage model peptides groups （ No. 7 - 10 ） . The quantitative expression was 0. 28, 0. 26, 0. 46 and 0. 30 respectively versus the negative control group. The relative expression of CTGF decreased in phage model peptides groups （ No. 7 - 10）. The quantitative expression was 0. 26, 0. 60, 0. 34 and 0. 17 respectively versus the negative control group. Conclusion Four phage model peptides （ No. 7 - 10） isolated from phage display 12-mer peptide library can inhibit the proliferation of keloid fibroblasts via regulating the expressions of NF-κB and CTGF.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2011年第38期2714-2718,共5页
National Medical Journal of China
基金
基金项目：国家自然科学基金（30670571,81071560）
山东省自然科学基金（ZR2010HQ010）
关键词
瘢痕疙瘩
成纤维细胞
肽库
转化生长因子Β1
细胞增殖
Keloid
Fibroblasts
Peptide library
Transforming growth factor-beta 1
Cellproliferation
