ABSTRACT
We have established a human umbilical vein endothelial cell (HUVEC) line monoclonal cells with the stable expression of human tissue-type plasminogen activator (t-PA) gene to provide a basis for further study on the vascular tissue engineering. Recombinant plasmid pcDNA3. 1-Myc-His B (-)/t-PA was constructed by insertion of t-PAcDNA originated from PBS/t-PA into eukaryotic expression vector pcDNA3. 1-Myc-His B(-) and transfected into hUVEC line cells mediated by lipofectamine. The positive clones were obtained by the screen of G418. The transcription and expression of t-PA gene were investigated by RT-PCR and Western blotting respectively. The t-PA activity was measured by chromogenic substrate assay. The positive clone cells which transcripted the mRNA of t-PA gene was obtained by RT-PCR. Immunoreactive human t-PA of the medium was significantly increased in the group of transfected gene when compared with that in the controlled and transfected plasmid without t-PA gene group. The biological activity of the protein of the t-PA in the media was increased significantly in the positive clone cells with t-PA gene transfected. The HUVEC line monoclonal cells with the stable expression of t-PA gene was established successfully.
Subject(s)
Humans , Cell Line , Human Umbilical Vein Endothelial Cells , Metabolism , Lipids , Pharmacology , RNA, Messenger , Genetics , Recombinant Proteins , Genetics , Tissue Engineering , Tissue Plasminogen Activator , Genetics , TransfectionABSTRACT
Objective To investigate the in vivo transduction capability of fusion protein PEP-1-EGFP with mice.Methods Two prokaryotic expression plasmids pET15b-EGFP and pET15b-PEP-1-EGFP were constructed and transformed into E.coli BL21(DE3) to express EGFP and fusion protein PEP-1-EGFP,respectively.The expressed EGFP and PEP-1-EGFP were purified with Ni2+-resin affinity chromatography.Five hundred micrograms of EGFP and PEP-1-EGFP fusion protein were injected into mouse through caudal vein,respectively,the mice were euthanized and perfused with PBS 2 hours after administration.Then,the heart,brain,liver,spleen and kidney were removed and sectioned with a cryostat at 7 ?m for visualization with a inverted fluorescent microscope.ResultsThe brain,heart,liver,spleen and kidney injected with PEP-1-EGFP showed bright and homogenous green fluorescence whereas that with EGFP showed no green fluorescence at all.Conclusion The successful expression and purification of PEP-1-EGFP fusion protein and its efficient transduction into mice in vivo provide a basis for the research on transmembrane delivery of macromolecule drugs mediated by the cell-penetrating peptide,PEP-1.
ABSTRACT
BACKGROUND: The incidence rate of colon cancer is on an obvious increase. Previous treatments are primarily surgical therapy; radiotherapy and chemotherapy. Gene therapy has been applied in the clinical practice for treating colon cancer.OBJECTIVE: To construct replication deficient hp27mt recombinant adenovirus and detect its expression in SW480 cell in order to investigate the viability of gene therapy based on adenovirus and study the anti-tumor characteristic of mutant p27.DESIGN: A non-randomized controlled trial.SETTING: Department of Gastroenterology, Affiliated People' s Hospital of Yunyang Medical College, Institute of Clinical Medical Science of Yunyang Medical College; Department of Gastroenterology, Zhongnan Hospital of Wuhan University.MATERIALS: The experiment was conducted in the Institute of Clinical Medicine, Yunyang Medical College from January 2002 to September 2003. Restriction endo-nucleases Age Ⅰ, Nhe Ⅰ, Kpn Ⅰ, Pac Ⅰ and Pme Ⅰ;Taq polymerase; T4 DNA ligase; Western blot kit; mouse anti-human p27kipl polyclonal antibody, horseradish-peroxidase conjugated goat anti mouse second IgG monoclonal antibody; pORF9-hp27mt plasmid, adenovirus framework plasmid pAdeasy-1, shuttle plasmid pShuttle-CMV, LacZ recombinant adenovirus Ad-LacZ, liposome, colon cancer cell line SW480.METHODS: hp27mt was excised from vector pORF9-hp27mt by restriction endonucleases, and inserted into shuttle plasmid pShuttle-CMV after two cycles of subclone, forming plasmid pShuttle-CMV-hp27mt. Then it was transformed by linear terminus(cut by PmeI) plasmid pShuttle-CMV-hp27mt into competent E. coli BJ5183 which contained adenovirus framework plasmid pAdeasy-1 to ensure that homologous recombination occurred. After correct identification of the transformant, it was digested by PacⅠ and transfected Ad293 cells, and packed into recombinant adenovirus Ad-hp27mt. Recombinant adenovirus was identified by polymerase chain reColon cancer cell line SW480 was infected with recombinant adenovirus Ad-p27mt, and expression of p27 protein was detected by Western blot.of expressed p27 protein after Ad-p27mt transfected colon cancer cell.novirus framework plasmid pAdeasy-1 with pShuttle-CMV-hp27mt, 30%combinant adenovirus DNA contained the target gene. Virus titer of the recancer cells, p27 was highly expressed in SW480 cells, as compared to low expression in non-transfected cells and Ad-LacZ transfected cells.CONCLUSION: Adenovirus, a type of gene transferring vector, can efficiently mediate p27 expression in tumor cells, which provides effective gene transfecting carrier for treating colorectal cancer with mutation p27 gene.
ABSTRACT
AIM: To study the inducing effect of human mutant p27 gene on apoptosis of the colorectal cancer cells. METHODS: The recombinant adenovirus Ad-p27mt was constructed to infect the colorectal cancer cell SW480. The inducing effect of Ad-p27mt on apoptosis in colorectal cancer cells was measured by flow cytometry, DNA fragment analysis and TUNEL method. RESULTS: Ad-p27mt was successfully constructed. When the multiplicity of infection (MOI) was ≥50, the infection efficiency reached 100%. After 24 h of infection, there was an apoptotic hypodiploid peak observed by flow cytometry before G_1 and there were apoptotic characteristic bands in the DNA electrophoresis. The apoptotic index detected by TUNEL method was 82.6?3.2 (Ad-p27mt group) and 5.0?3.5 (control group), respectively, the difference of which was significant (P