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• 札幌医科大学   医学部 医学科 口腔外科学講座 医学部医学科臨床医学部門講座口腔外科学講座   助手  

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• 口腔外科学

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• Indian hedgehog gene transfer augments hematopoietic support of human stromal cells including NOD/SCID-beta(2)m(-/-) repopulating cells

  M Kobune, Y Ito, Y Kawano, K Sasaki, H Uchida, K Nakamura, H Dehari, H Chiba, R Takimoto, T Matsunaga, T Terui, J Kato, Y Niitsu, H Hamada

  BLOOD ( AMER SOC HEMATOLOGY )  104 ( 4 ) 1002 - 1009  2004年08月

  　概要を見る

  Hematopoietic stem cells (HSCs) are a subset of bone marrow cells that are capable of self-renewal and of giving rise to all types of blood cells. However, the mechanisms involved in controlling the number and abilities of HSCs remain largely unknown. The Indian hedgehog (Ihh) signal has an essential role in inducing hematopoietic tissue during embryogenesis. We investigated the roles of the Ihh in coculture with CD34(+) cells and human stromal cells. Ihh mRNA was expressed in primary and telomerized human (hTERT) stromal cells, and its receptor molecules were detected in CD34+ cells. Ihh gene transfer into hTERT stromal cells enhanced their hematopoietic supporting potential, which was elevated compared with control stromal cells, as indicated by the colony-forming units in culture (CFU-Cs) (26-fold 2-fold versus 59-fold +/- 3-fold of the initial cell number; mixed colony-forming units [CFU-Mix's], 63-fold +/- 37-fold versus 349-fold +/- 116-fold). Engraftments of nonobese diabetic/severe combined immunodeficiency-beta(2)m(-/-) (NOD/SCID-beta(2)m(-/-)) repopulating cells (RCs) expanded on Ihh stromal cells were significantly higher compared with control coculture results, and engraftment was neutralized by addition of an antihedgehog antibody. Limiting dilution analysis indicated that NOD/SCID-beta(2)m(-/-) RCs proliferated efficiently on lhh stromal cells, compared with control stromal cells. These results indicate that lhh gene transfer could enhance the primitive hematopoietic support ability of human stromal cells. (C) 2004 by The American Society of Hematology.

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• Adenovirus-mediated transfer of siRNA against survivin induced apoptosis and attenuated tumor cell growth in vitro and in vivo

  H Uchida, T Tanaka, K Sasaki, K Kato, H Dehari, Y Ito, M Kobune, M Miyagishi, K Taira, H Tahara, H Hamada

  MOLECULAR THERAPY ( ACADEMIC PRESS INC ELSEVIER SCIENCE )  10 ( 1 ) 162 - 171  2004年07月

  　概要を見る

  Gene targeting using short interfering RNA (siRNA) has become a common strategy to explore gene function because of its prominent efficacy and specificity. For the application of siRNA technology to gene therapy, however, still more efficient transduction of siRNA into target cells is needed. In this study, we developed an adenoviral vector harboring a tandem-type siRNA expression unit, in which sense and antisense strands composing the siRNA duplex were separately transcribed by two human U6 promoters. Targeting survivin, an antiapoptotic molecule widely overexpressed in malignancies but not detected in terminally differentiated adult tissues, this type of adenoviral vector (Adv-siSurv) successfully exerted a gene knockdown effect and induced apoptosis in HeLa, U251, and MCF-7 cells. These cancer cells, once infected with Adv-siSurv, displayed remarkably attenuated growth potential, both in vitro and in vivo. Moreover, intratumoral injection of Adv-siSurv significantly suppressed tumor growth in a xenograft model using U251 glioma cells. This novel modality may be a promising tool for cancer therapy.

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• Pre-administration of angiopoietin-1 followed by VEGF induces functional and mature vascular formation in a rabbit ischemic model

  A Yamauchi, Y Ito, M Morikawa, M Kobune, JH Huang, K Sasaki, K Takahashi, K Nakamura, H Dehari, Y Niitsu, T Abe, H Hamada

  JOURNAL OF GENE MEDICINE ( JOHN WILEY & SONS LTD )  5 ( 11 ) 994 - 1004  2003年11月

  　概要を見る

  Background Angiopoietin-1 (Ang1) and vascular endothelial growth factor (VEGF) play important roles in vascular formation and maturation, suggesting that the combination of these two would be a promising therapy for ischemia. However, it remains unclear what the best schedule of administration of these cytokines might be. Methods Six experimental groups were used to prepare the rabbit ischemic hindlimb model following naked plasmid intramuscular administration as follows: empty vector (C), single gene (Ang1, A; VEGF, V), Ang-1 followed by VEGF (A - V), co-administration of Ang1 and VEGF (A + V), and VEGF followed by Ang1 (V - A). Results Thirty days after gene administration, A - V showed a significantly increased blood pressure and blood-flow recovery in the ischemic limb compared with the control group. Histological findings by alpha-smooth muscle-actin (alpha-SNM) staining revealed that the two combination groups had more mature vessels as compared with the control group. Significantly, A - V revealed the highest density of alpha-SMA-positive vessels compared with VEGF alone or Ang1 alone. Angiographic assessment revealed that A - V had a greater increased arterial diameter compared with VEGF alone. Edema, one of the major adverse effects induced by VEGF, was not found in A - V throughout the experiments, while VEGF alone and V - A showed severe edema induced by VEGF. Conclusions The pre-administration of Ang1 followed by VEGF resulted in an improvement of hemodynamic status, an increased number of vessels covered with a-actin-positive mural cells, and prevention of VEGF-mediated edema. Thus, priming by Ang1 gene administration would be beneficial for therapeutic angiogenesis in VEGF gene therapy. Copyright (C) 2003 John Wiley Sons, Ltd.

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• Adenoviral-delivered angiopoietin-1 reduces the infarction and attenuates the progression of cardiac dysfunction in the rat model of acute myocardial infarction

  K Takahashi, Y Ito, M Morikawa, M Kobune, JH Huang, M Tsukamoto, K Sasaki, K Nakamura, H Dehari, K Ikeda, H Uchida, S Hirai, T Abe, H Hamada

  MOLECULAR THERAPY ( ACADEMIC PRESS INC ELSEVIER SCIENCE )  8 ( 4 ) 584 - 592  2003年10月

  　概要を見る

  In acute myocardial infarction (AMI), prognosis and mortality rate are closely related to the infarct size and the progression of postinfarction cardiac failure. Angiogenic gene therapy has presented a new approach for the treatment of AMI. Angiopoietin-1 (Ang1) is a critical angiogenic factor for vascular maturation and enhances vascular endothelial growth factor (VEGF)-induced angiogenesis in a complementary manner. We hypothesized that gene therapy using Ang1 for AMI might promote angiogenesis cooperatively with intrinsic VEGF, since high concentrations of circulating VEGF have been reported in AMI. To evaluate our hypothesis, we employed a rat AMI model and adenoviral Ang1 (HGMW-approved gene symbol ANGPT1) gene transfer to the heart. A significant increase in capillary density and reduction in infarct sizes were noted in the infarcted hearts with adenoviral Ang1 gene treatment compared with control infarcted hearts treated with saline or adenoviral vector containing the beta-galactosidase gene. Furthermore, the Ang1 group showed significantly higher cardiac performance in echocardiography (55.0% of ejection fraction, P < 0.05 vs control) than the saline or adenoviral controls (36.0 or 40.5%, respectively) 4 weeks after myocardial infarction. The adenoviral delivery of Ang1 during the acute phase of myocardial infarction would be feasible to attenuate the progression of cardiac dysfunction in the rat model.

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• Myocardial injection of CA promoter-based plasmid mediates efficient transgene expression in rat heart

  JH Huang, Y Ito, M Kobune, K Sasaki, K Nakamura, H Dehari, K Takahashi, K Ikeda, H Uchida, K Kato, H Hamada

  JOURNAL OF GENE MEDICINE ( JOHN WILEY & SONS LTD )  5 ( 10 ) 900 - 908  2003年10月

  　概要を見る

  Background Although naked plasmid injection is the safest and most convenient method for gene delivery, a major limitation of this approach is currently poor transgene expression. The CA promoter (chicken beta-actin promoter with cytomegalovirus, CMV, enhancer) is one of the strongest transcriptional control modules found; however, it is uncertain whether a CA promoter-based vector is efficient enough for naked gene therapy in a cardiovascular context. Methods The beta-galactosidase (LacZ) expression provided by CA promoter plasmid (pCAZ2) injection into the skeletal muscle or the heart of Lewis rats was compared with CMV promoter plasmid or adenoviral vector (AxCAZ3). The effect of Simian virus 40 of the replication origin (SV40ori) deletion from pCAZ2 on transgene expression was also evaluated. Results pCAZ2 showed the highest LacZ expression in both skeletal muscle and heart in comparison with the CMV promoter-based vector 5 days after naked plasmid injection. LacZ expression in the heart obtained using 20 mug of pCAZ2 was almost equivalent to that shown with AxCAZ3 at 6.0 x 10(9) optical particle units. The time course of transgene expression driven by CMV and CA promoters in the heart were similar, with the CA promoter providing significantly higher gene expression than the CMV promoter across all time points examined. SV40ori deletion from pCAZ2 did not affect transgene expression in either skeletal muscle or heart. Conclusions Transgene expression mediated by naked CA promoter-based plasmid injection was shown to be quite efficient in the heart. We propose that the CA promoter vector is suitable for myocardial gene therapy. Copyright (C) 2003 John Wiley Sons, Ltd.

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