Biomedical subjects

Teru Okitsu

Publications and source records attributed to Teru Okitsu.

39 records · Page 3Linked to original sources

PubMedDate not supplied

Improvement in the differentiated hepatic phenotype of immortalized human hepatocytes by adenovirus mediated p21 gene transfer.

The p21 molecule, a potent cyclin dependent kinase inhibitor, regulates the transition from the G1 phase to the S phase of the cell cycle and is involved in terminal cellular differentiation. The overexpression of p21 has been shown to induce differentiation in various cell lines. We have made an effort to establish a reliable human hepatocyte cell line as a source of hepatic function in bioartificial liver (BAL) therapy. In this work, we investigated the effect of p21 on the differential phenotype of simian virus 40 large T antigen (SV40Tag) immortalized human hepatocytic NKNT-3 cells. A recombinant adenoviral vector expressing a p21 gene under control of the cytomegalovirus (CMV) promoter (Ad-p21) was used to efficiently transfer genes into NKNT-3 cells. The morphologic alterations, the cell cycle progression, and the expression of p-450 associated enzymes (CYPs) were carefully examined in NKNT-3 cells that had been infected with Ad-p21. Adenovirus mediated gene delivery of p21 was efficiently achieved in NKNT-3 cells without affecting cellular structure. After Ad-p21 infection, NKNT-3 cells were G0/G1 arrested in cell cycle analysis. NKNT-3 cells that had been infected with Ad-p21 showed differentiated hepatic phenotypes in morphology and improvement in protein expression of CYP 3A4 and CYP 2C9. In the present work, we demonstrate that the exogenous expression of p21 enhances the differential phenotype of immortalized hepatocytic NKNT-3 cells.

Adenoviridae↗

PubMedDate not supplied

Lentiviral vector: a useful tool for transduction of human liver endothelial cells.

Endothelial cells play multiple roles in pathophysiologic processes and are increasingly being recognized as target cells of gene therapy. Lentiviral vectors derived from human immunodeficiency virus type 1 have an ability to infect both dividing and nondividing cells and currently receive a great deal of attention as an innovative tool for transduction of target cells. The purpose of the present work was to evaluate the efficacy of a lentiviral vector for transducing human liver endothelial cells (HLECs) in vitro. For the present study, a pseudotyped lentiviral vector encoding a green fluorescent protein (GFP) gene, LtV-GFP, was generated by means of FuGENE 6 method and allowed to infect HLECs. Approximately 95% of HLECs were positive for GFP expression after LtV-GFP infection at a multiplicity of infection of 10. Notably, LtV-GFP transduced HLECs had stable and long term GFP expression, showed gene expression of endothelial markers including CD 34, factor VIII, flt-1, KDR/flk-1 and HGF, and maintained in vitro angiogenic potential in a Matrigel assay to the same extent as primarily cultured HLECs. These findings provide evidence that lentivirus based gene delivery is an efficient tool for transduction of endothelial cells that could be considered for cell and gene therapies and hybrid artificial organs.

Biocompatible Materials↗

PubMedDate not supplied

Adenovirus mediated gene transduction of primarily isolated mouse islets.

Expansion of pancreatic islet cell populations, especially the beta cells, using a currently available ex vivo gene transfer technology is important to develop cell therapies to treat Type I diabetes. In this study, we evaluated adenovirus mediated gene transfer efficiency in primarily isolated mouse islet cells in two types of culture conditions: freshly isolated suspended islets and cultured islets with monolayer formation. A recombinant replication deficient adenovirus vector encoding a green fluorescence protein (GFP) cDNA, Ad/ CMV-GFP, was used in the present transduction experiments. Rat 804G derived extracellular matrix (804G-ECM) and 3-isobutyl-1-methylxanthine (IBMX) were used to facilitate monolayer formation of the isolated mouse pancreatic islets. Suspended islets were transfected with Ad/CMV-GFP at more than 95% efficiency. However, analysis of immunohistochemical stains for insulin and glucagon in thin sliced sections of the islets revealed that GFP expression was localized just in the outer cells of the islets, almost all of which were glucagon positive alpha-cells. The beta-cells existing at the inner area of the suspended islets were GFP negative. In contrast, under the condition of the islets in monolayer formation cultures, all of the islet cells including the beta-cells were efficiently infected with Ad/CMV-GFP. To achieve an efficient adenoviral gene transfer to the pancreatic beta-cells, monolayer formation of the islets is critical. Such culture conditions were facilitated by combining 804G-ECM with IBMX.

1-Methyl-3-isobutylxanthine↗