| Project/Area Number |
06454274
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Respiratory organ internal medicine
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| Research Institution | Juntendo University School of Medicine |
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Principal Investigator |
DAMBARA Takashi Juntendo University School of Medicine, Respir Med. Associate Prof, 医学部, 助教授 (30102263)
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| Co-Investigator(Kenkyū-buntansha) |
HASUNUMA Kiichi Juntendo University School of Medicine, Respir Med, Assistant Prof, 医学部, 講師 (10208473)
OKA Masahiko Juntendo University School of Medicine, Respir Med, Research Associate, 医学部, 助手 (60203965)
SETOGUCHI Yasuhiro Juntendo University School of Medicine, Respir Med, Research Associate, 医学部, 助手 (90206649)
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| Project Period (FY) |
1994 – 1995
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| Project Status |
Completed (Fiscal Year 1995)
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| Budget Amount *help |
¥5,100,000 (Direct Cost: ¥5,100,000)
Fiscal Year 1995: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1994: ¥3,300,000 (Direct Cost: ¥3,300,000)
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| Keywords | nitric oxide / nitric oxide synthase / gene transter / pulmonary arterial hypertension / endothelium / smooth muscle / E.coli Lac Z gene / プラスミドベクター / RT-PCR / cGMP |
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| Research Abstract |
1. Endothelial derived-nitric oxide synthase (eNOS) was isolated from cDNA library of human umbilical vein.
2. Two type of plasmid vector were constructed : pCMVeNOS containing eNOS cDNA driven by cytomegalovirus promoter ; pCMVLacZ containing E.coli LacZ gene driven by the same promoter.
In vitro gene transfer
1. To evaluate the efficacy of plasmid DNA-mediated gene transfer to bronchial epithelial cells (BE), pulmonary arterial endothelial cells (Pend) and pulmonary arterial smooth muscle cells (Psm), pCMVLacZ was administered to the cultured BE, Pend and Psm. 48 hours after administration of pCMVLacZ, X-Gal staining showed blue coloration of the BE, Pend and Psm, indicative beta-galactosidase activity. The proportion of BE, Pend and PSm with beta-galactosidase activity revealed 0.24%, 0.18% and 0.11% respectively.
2. To evaluate the expression of eNOS following transfer of eNOS cDNA, BE, Pend and Psm were treated with pCMVeNOS.Immunohistochemical staining using anti-human eNOS monoclona
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l antibody revealed eNOS expression. To assess the function of eNOS expressed on BE,Pend and Psm following transfer of eNOS cDNA,nitric oxide (NO) in the culture medium on each type of cells was measured by using Sievers 270B.NO in the culture medium on BE, Pend or Psm following transfer of eNOS cDNA averaged 6 fold production of the base line. Furthermore, Psm received eNOS cDNA resulted in slow proliferation-rate compared to control Psm.
In vivo gene transfer
1. To evaluate in vivo plasmid DNA-mediated gene transfer to lung, pCMVLacZ was administered on intratracheal approach or intravenous approach via pulmonary artery using catheter. Lung from rats receiving intratrachal administration of pCMVLacZ revealed blue coloration on small number of bronchial epithelial cells. In contrast, Pulmonary artery from rats receiving intravenous administration of pCMVLacZ revealed of endothelial cells.
2. Based on these results, pCMVeNOS was administered to air-way. Immunohistochemical staining showed eNOS expression on BE from rats following intratracheal administration of pCMVeNOS.However, no effect to pulmonary vasomotor function was evident in measurement of pulmonary arterial pressure under the exposure of Angiotensin II. Less
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