合成角质细胞生长因子活性短肽促进表皮细胞增殖的实验研究  

The Experiment of Keratinocyte Growth Factor Bioactive Peptides' Synthesis and Promoting Epidermal Cell Proliferation

作　　者：宗宪磊[1] 陆海滨[1] 祁佐良[1] 李国菊[2] 宋国栋[1] 都乐[1] 靳小雷[1] 姜笃银[3] 

机构地区：[1]中国医学科学院北京协和医学院整形外科医院,北京市100144 [2]山东大学口腔医学院放射科,山东省济南市250014 [3]山东大学第二医院美容整形烧伤外科、急诊科,山东大学组织工程研究所,山东省济南市250033

出　　处：《组织工程与重建外科杂志》2016年第1期1-5,共5页Journal of Tissue Engineering and Reconstructive Surgery

基　　金：国家自然科学基金项目(30670571,81201467);山东省自然科学基金项目(ZR2011HM027)

摘　　要：目的应用噬菌体随机7肽库筛选角质细胞生长因子(Keratinocyte growth factor,KGF)的关键序列,进行KGF活性短肽的调整和合成,并研究其对表皮细胞增殖的作用。方法以KGF单克隆抗体为靶,筛选噬菌体随机7肽库,获得KGF的特异性序列,并进行序列调整。用免疫荧光法检测KGF活性短肽的表皮细胞亲和力。用CCK-8法检测KGF活性短肽对表皮细胞增殖的影响。用RT-PCR法和Western-blot法检测表皮细胞上特异性受体KGFR的表达水平。结果筛选噬菌体随机7肽库,获得2个与KGF相似的特异性序列,合成获得2个KGF活性短肽,并纯化至98%纯度,短肽的C端进行氨基化封闭,N端进行罗丹明激光染料标记。免疫荧光检测结果显示,2个KGF活性短肽均能够与表皮细胞相结合。CCK-8检测结果显示,与阴性对照组相比,2个KGF活性短肽能够促进表皮细胞增殖,并呈浓度依赖性。RT-PCR和Western-blot检测结果显示,2个KGF活性短肽组中表皮细胞表达KGFR增强。结论从噬菌体随机7肽库中筛选到2个KGF的关键序列,合成的2个KGF活性短肽能够与KGFR相结合,并增强KGFR的表达,从而促进表皮细胞增殖,有望应用于促进创面愈合。Objective To isolate sequences from a phage display 7-mer peptide library, synthesize keratinocyte growth factor（KGF） bioactive peptide from the isolated sequences and to evaluate the effects of KGF bioactive peptide on epidermal cell proliferation. Methods Using monoclonal anti-human KGF antibody as the target, a phage display 7-mer peptide library was screened and target sequences were isolated. The sequences were then modified to generate KGF bioactive peptides. Immunofluorescence assay was performed to evaluate the binding activity of KGF peptides on epidermal cells.CCK-8 was used to detect the effects of KGF bioactive peptide on epidermal cell proliferation. RT-PCT and Western-blot were used to detect the expression of KGF receptor of epithelial cells. Results Two peptides similar to KGF was isolated from a phage display 7-mer peptide library and then modified to generate two KGF bioactive peptides. KGF bioactive peptides were then purified to reach a purity of 98%. The C terminal of KGF bioactive peptides was closed, and the N terminal was labeled with rhodamine dye laser. KGF bioactive peptides were able to combine with the epidermal cells showed by Immunofluorescence assay. CCK-8 test results showed that compared with the control group, the two KGF bioactive peptides promoted epidermal cell proliferation in a dose-dependent manner. RT-PCR and Western-blot test results showed two KGF bioactive peptidegroup enhanced the expression of KGFR on epidermal cells. Conclusion KGF bioactive peptides can be synthesized based on sequences screened from phage display peptide library. KGF bioactive peptides are capable of combining with KFGR, enhancing the expression KGFR, and thereby promoting the proliferation of epidermal cells. The results are expected to help promoting wound healing.

关 键 词：噬菌体随机7肽库 角质细胞生长因子 活性短肽 表皮细胞 创面愈合 

分 类 号：R752.53[医药卫生—皮肤病学与性病学]