| Project/Area Number |
08457284
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Kidney internal medicine
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| Research Institution | School of Medicine Tokai University |
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Principal Investigator |
KUROKAWA Kiyosi School of Medicine Tokai University Nephrology & Metabolism Div.Proffessor, 医学部, 教授 (30167390)
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| Co-Investigator(Kenkyū-buntansha) |
FUKAGAWA Masafumi University of Tokyo Faculty of Medicine First Department of Internal Medicine Re, 医学部, 医員 (00211516)
MIYATA Tosio School of Medicine Tokai University Inst.of Medical Science & Department of Medi, 総合医学研究所, 講師 (10222332)
飛田 美穂 東海大学, 医学部, 助教授 (20147169)
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| Project Period (FY) |
1996 – 1997
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| Project Status |
Completed (Fiscal Year 1997)
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| Budget Amount *help |
¥7,000,000 (Direct Cost: ¥7,000,000)
Fiscal Year 1997: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1996: ¥5,700,000 (Direct Cost: ¥5,700,000)
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| Keywords | parathyroid / parathyroid hyperplasia / cell prolifiration / PTH / vitamin D / vitamin D receptor / adonovirus / gene therapy / ビタミンD, / ビタミンD受容体, / カルシウムセンサー, / 過形成 / 腎不全 / 核増殖抗原 / アデノウイルススベクター |
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| Research Abstract |
Recent data suggest that deranged gene reguration underlie the parathy-roid dysfunction in chronic failure. However, the mechanism of this abnormality has not been fully elucidated at cellular level because no estsblished parathyroid cell line is practically available.
To modulate funnction of diapersed parathyroid cells which maintain physiological response to extracellular only for a few days, we transfered functional genes by adenovirus vector.
1.Analysis of Vitamin D Receptor (VDR) Density and Cell Prolifiration in Parathyroid Hyperplasia.
Immunohistochemistry of serial section of surgically exsized parathyroid glands from uremic patients revealed that positive rate of VDR and proliferating cell nuclear antigen (PCNA) had significant negative coreration. Further more, cells in small nodule forming within diffuse parathyroid hyperplasia had low density of VDR and higher positive rate of PCNA compared with surrounding cells. These data suggest more direct coreration between decreased de
… More
nsity of VDR and cell proliferation in chronic renal failure.
2.Gene Transfer into Parathyroid Cells by Adenovirus
Surgically excised parathyroid glands were dispersed by collagenase and 5x105cells in 0.5ml of DMEM/F12 (FBS10%) were cultured in each well of 24-well dish. On the next day, replication-deficient adenoviral vector that contained a reporter gene enconding thenuclear b-galactosidase driven by CAG promoter was added at 1x107pfu/ml or less. X-gal staining was positive in almost all cells at 48hours of infection. At this point, adenovirus infection itsself did not affect the cell number BrdU incorporation. Basal and phisiolosical response of PTH secretionto low and high calcium ion concentration were not affected by viral infection either. Thus, successful gene transfer was accomplished by adenoviral vectors in parathyroid cells which still maintained physiological response to calcium.
Furthermore, we could also successfully infect parathyroid cells in vivo by directly injecting virus solution. Next we constracted recommbinant adenovirus which expresses human VDR gene under CAG promoter. Trnsfer of VDR gene into dispersed parathyroid cells by this virus lead to significant suppression of PTH secretion in the presense of 1/10 of physiological concentration of calcitoriol.
Thus, it may soon become possible to modulate parathyroid function in vivo by trnsferring functional genes by adenovirus vector. Less
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