RESUMO
PURPOSE: To construct an EGF receptor (EGF-R)-mediated histone H1(0)-based gene delivery system for gene therapy. METHODS: A recombinant DNA containing histone H1(0), EGF-R ligand, and endosomalytic domains was constructed in a prokaryotic vector and expressed in E. coli. Expression of the beta-galactosidase (beta-gal) gene in the tumor cells and tissues was observed after transduction of the beta-gal gene packaged by purified fusion proteins in vitro and in vivo. RESULTS: As an extension of the research on previously reported chemically synthetic composite polypeptide gene delivery systems, this genetically engineered polypeptide has proved to be capable of targeting the beta-galactosidase (beta-gal) gene into EGF-R-positive cancer cells both in vitro and in vivo. We also studied the time course of beta-gal gene expression in tumor tissues delivered in vivo by this polypeptide vector. At 24 h after administration, expression of the beta-galactosidase gene in tumor reached peak levels. The dosage optimization of administered polyplex was also investigated. The optimal dose of polyplex per mouse was 1 microg DNA packaged by 3 microg of composite polypeptide. CONCLUSIONS: The genetically engineered polypeptide based on histone H1(0) is a promising gene delivery system targeting EGF-R.
Assuntos
Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/terapia , Receptores ErbB/metabolismo , Terapia Genética/métodos , Histonas/genética , Osteossarcoma/metabolismo , Osteossarcoma/terapia , Animais , Neoplasias Ósseas/enzimologia , Neoplasias Ósseas/genética , Escherichia coli , Feminino , Técnicas de Transferência de Genes , Vetores Genéticos , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Proteínas de Fusão Oncogênica/análise , Proteínas de Fusão Oncogênica/isolamento & purificação , Osteossarcoma/enzimologia , Osteossarcoma/genética , Plasmídeos/genética , Reação em Cadeia da Polimerase , Células Tumorais Cultivadas , beta-Galactosidase/metabolismoRESUMO
OBJECTIVE: To investigate gene transfer efficiency of a novel target non-viral vector GE7 and effects of herpes simplex virus thymidine kinase (HSV(1)-tk)/ganciclovir (GCV) mediated by it in vitro. METHODS: The epidermal growth factor receptor (EGF-R) target gene delivery system GE7 was constructed. Human ovarian cancer cell line CAOV3 was transfected in vitro with beta-galactosidase (beta-gal) as reporter gene and HSV(1)-tk gene as therapeutic gene using this gene delivery system. By means of the assay of X-gal staining, Northern blotting, cell growth-inhibiting curve and so on, the transferring efficiency of exogenous genes and killing effects are observed. RESULTS: It showed that gene transfer efficiency is over 80%. When 10 mg/L GCV was put into ovarian cells transfected with HSV(1)-tk gene, 95% of cells were killed, and the apoptosis ratio reached up to 30. CONCLUSIONS: The GE7 gene delivery system is an effective and safe delivery system. GE7/HSV(1)-tk/GCV therapeutic gene system is appraising for ovarian cancer.
Assuntos
Receptores ErbB/genética , Terapia Genética/métodos , Neoplasias Ovarianas/terapia , Simplexvirus/enzimologia , Timidina Quinase/genética , Divisão Celular , Feminino , Citometria de Fluxo , Ganciclovir/uso terapêutico , Vetores Genéticos , HumanosRESUMO
Histone H1 contributes to condense nucleosome into super-structure during the transformation of chromatin into chromosome. It is shown in this rep or t that the fusion protein HNHG with the core of C-terminus of histone H1(0) expressed in BL21 (DE3) could also condense the plasmid DNA, just as histones did in nucleus. Under electron microscope, plasmid DNA condensed and supercoiled after t he addition of HNHG, in contrast to plasmid DNA control. This specific ability of the fusion protein HNHG of binding and condensing plasmids could be utilized to construct novel exogenous gene delivery systems. HNHG would be a promising candidate for gene delivery.
