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

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.

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.

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.
Cell Line ; Human Umbilical Vein Endothelial Cells ; metabolism ; Humans ; Lipids ; pharmacology ; RNA, Messenger ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Tissue Engineering ; Tissue Plasminogen Activator ; biosynthesis ; genetics ; Transfection