| Project/Area Number |
04454189
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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 |
Experimental pathology
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| Research Institution | Oita Medical University |
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Principal Investigator |
HIGUCHI Yasunori Oita Medical Univ., Pathology Professor, 医学部, 助教授 (60040284)
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| Co-Investigator(Kenkyū-buntansha) |
瀬戸口 美保子 大分医科大学, 医学部, 助手 (20236110)
AKIZUKI Shin'ichiro Oita Medical Univ., Pathology Assistant (80159334)
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| Project Period (FY) |
1992 – 1993
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| Project Status |
Completed (Fiscal Year 1993)
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| Budget Amount *help |
¥6,200,000 (Direct Cost: ¥6,200,000)
Fiscal Year 1993: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1992: ¥4,200,000 (Direct Cost: ¥4,200,000)
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| Keywords | CD14 / TNF / transgene / recombinant / diabetes / beta-cell / cDNA / beta-cell / 〓DNA / transgenic mouse / TNF-α / shock |
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| Research Abstract |
I.We analyzed the structure of the 5' end of the mouse CD14 (mCD14) gene. Sequentially 5'-deleted, chimeric and point mutated clones were tested for their ability to stimulate chroramphnicol acetyltransferase (CAT). An 8-base pair sequence, TGATTCAC, at position -255 enhanced the expression of the CAT gene in macrophage cell lines. The 5' upstream of the human CD14 gene were sequenced and compared with that of mouse. II.Recombinant mCD14 protein was deleted at carboxyterminus was produced in E.coli. We made monoclonal antibodies (mAb) against the protein. Immunohistochemical staining using mAb showed that CD14-positive Kupffer cells (KC) were small in number in the liver in nonstimulated mice. The number of stained KC gradually increased with time after intraperitoneal injection of LPS, peaked 6 h after injection, and returned to normal by 20 h after injection. Slight increase in mCD14 expression was observed in peritoneal macrophages 2 h after LPS administration in vivo using flow cytometric analysis. Unique features of mCD14 expression in KC were also confirmed by the expression of mCD14 mRNA which peaked at 3 h after LPS administration. Mice bearing a mCD14 transgene controlled by an metallotionein promoter (MTTG) were produced. Northen blot analysis of MTTG showed that the mice strongly expressed mCD14 mRNA in the liver, small intestine and testis. III.Mice bearing a TNFalpha transgene controlled by an insulin promoter developed an increasingly severe insulitis. Diabetes never divelopped, even though a number of potentially inducing conditions were used, including the prolonged perfusion of TFNgamma and the permanent expression of a non-tolerogenic viral protein on beta cells.
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