摘要
目的人α防御素主要由中性粒细胞(HNP1、HNP2和HNP3)和小肠潘氏细胞(HD5和HD6)分泌。HNP1-3可以被作为肿瘤标志物,小剂量的α防御素可以促进肿瘤的增长,大剂量的α防御素可以裂解杀死肿瘤细胞。制备人防御素(human defensin-5,HD5或D5)、力达霉素辅基蛋白(lidamycin apoprotein,LDP)与EGFR配体寡肽(EGFR-directing ligand peptide,Ec)组合的融合蛋白Ec-LDP-D5,并初步探讨其抗胰腺癌活性。方法采用基因工程的方法制备融合蛋白基因,并将其表达纯化得到融合蛋白Ec-LDP-D5。融合蛋白与细胞表面EGFR的亲和活性采用ELISA和细胞免疫荧光化学法;采用CCK-8法测定融合蛋白对人胰腺癌细胞PNAC-1和Aspc-1的体外杀伤活性;流式细胞术检测Ec-LDP-D5蛋白对细胞凋亡的影响。结果成功构建并表达融合蛋白Ec-LDP-D5,产物主要以包涵体的形式存在,目的蛋白经纯化、复性后,每升发酵液可以获得1mg纯度达到85%。Ec-LDP-D5可与高表达EGFR的PNAC-1和Aspc-1细胞表面结合。Ec-LDP-D5对PNAC-1和Aspc-1肿瘤细胞有强烈的杀伤作用,Ec-LDP-D5蛋白对Aspc-1肿瘤细胞抑制率为76.8%,与Ec-LDP的抑制率49.4%相比,差异有统计学意义,P=0.038;对PANC-1细胞抑制率为64.6%,与Ec-LDP的抑制率28.3%相比,差异有统计学意义,P=0.042。Ec-LDP-D5在8μmol/L的浓度下即可强烈的诱导细胞发生凋亡,细胞凋亡率(24.6±0.56)%,与Ec-LDP的凋亡率相比P=0.011。结论本实验制备的防御素融合蛋白Ec-LDP-D5可以与高表达EGFR的PNAC-1和Aspc胰腺癌细胞结合,对胰腺癌细胞具有强烈的杀伤活性和诱导凋亡能力,具有发展为抗肿瘤靶向药物的潜能。
OBJECTIVE α-defensins are mainly packaged in neutrophil granules(HNP1,HNP2,HNP3)or secreted by intestinal Paneth cells(HD5,HD6).HNP1-3peptides are also secreted and their accumulation in biological fluids was proposed as a tumor biomarker.As reported,α-defensins promote tumour cell growth;however,provoke cell death at higher concentration.The aim of this study was to construct a novel fusion protein consisting of oligopeptides specific for human epidermal growth factor receptor(EGFR),lidamycin apoprotein(LDP)and human defensin 5(HD5),and to investigate its anti-pancreatic cancer activity.METHODS By DNA recombination,HD5 was fused to the protein Ec-LDP to obtain a fusion protein,Ec-LDP-D5.Immunofluorescence assay and ELISA assay were used to investigate the binding activity of Ec-LDP-D5 to EGFR overexpressed cancer cells.CCK-8assay was used to measure the in vitro cytotoxicity of Ec-LDP-D5 and AnnexinⅤ-FITC/PI staining assay was used to analyze its apoptosis-inducing efficacy.RESULTS Fusion protein Ec-LDP-D5 was constructed correctly and expressed in E.coli in a insoluble inclusion bodies.The production of Ec-LDP-D5 was 1mg per liter fermentation broth,and the purity of fusion protein was 85% as analyzed by HPLC.Ec-LDP-D5 showed strong binding activity to pancreatic cancer cells highly expressing EGFR,such as PNAC-1and Aspc-1cells.The fusion protein Ec-LDP-D5 exhibited more potent cytotoxicity to cancer cells as compared with Ec-LDP,respectively.The results from Annexin Ⅴ-FITC/PI staining assay also revealed that Ec-LDP-D5 significantly induced cell apoptosis.CONCLUSION The novel fusion protein Ec-LDP-D5 bounds to EGFR specifically,and shows potent cytotoxicity and apoptosis-inducing activity to pancreatic cancer cells,which suggests that it would be a promising candidate of cancer therapeutics.
出处
《中华肿瘤防治杂志》
CAS
北大核心
2016年第20期1344-1350,共7页
Chinese Journal of Cancer Prevention and Treatment
基金
国家自然科学基金(81502691)
山东省自然科学基金(ZR2015HQ011)
山东省医药卫生科技发展计划(2016WS0547)
关键词
胰腺肿瘤
融合蛋白
人表皮生因子受体
力达霉素辅基蛋白
人防御素5
pancreatic neoplasms
fusion protein
human epidermal growth factor receptor
lidamycin apoprotein
human defensin 5
