| Project/Area Number |
09670462
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
内科学一般
|
|---|
| Research Institution | HOKKAIDO UNIVERSITY |
|---|
Principal Investigator |
KOBAYASHI Seiichi Coll.of Med.Tech., Hokkaido Univ., Prof., 医療技術短期大学部, 教授 (30150246)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
KOIKE Takao School of Med., Hokkaido Univ., Prof., 医学部, 教授 (80146795)
|
|---|
| Project Period (FY) |
1997 – 1998
|
| Project Status |
Completed (Fiscal Year 1998)
|
| Budget Amount *help |
¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 1998: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1997: ¥1,700,000 (Direct Cost: ¥1,700,000)
|
|---|
| Keywords | systemic lupus erythematosus / soluble Fas / heterogeneity / fusion protein / apoptosis / monoclonal antibody / sandwich ELISA / gene transfer / 抑制機序 |
|---|
| Research Abstract |
Research results obtained from the project entitled "Analysis of soluble Fas molecules in systemic lupus erythematosus (SLE) and establishment of model animals", are as follows.
1. Several PCR products were obtained from the RT-PCR analysis of full-length Fas gene expression by peripheral blood mononuclear cells from SLE patients. Sequencing analysis revealed that PCR products were derived from membranous Fas (mFas) and tow soluble Fas mRNA which were exon 6-deficient and exons 3, 4, and 6-deficient (Fas*Ex6 and Fas*Ex3/4/6, respectively). Presumed N-terminal 49 amino acids encoded by exon 2 were common in these soluble Fas molecules, whereas others were different between soluble Fas molecules.
2. A recombinant fusion protein (FasEx2-Ig) consisting of amino acid encoded by Fas exon 2 and Fc protein of human IgG1 was purified to examine the in vitro function of soluble Fas. Fas Ex2-Ig inhibited Fas-dependent apoptosis as did FasExt-Ig by soluble Fas. FasEx-Ig inhibited Fas-dependent apotosis as vitro function soluble Fas. FasEx2-Ig by three mechanisms ; ligand competition, blocking of FasL binding to mFas and ineffective trimerization for death signal transduction.
3. Two mAbs were established to detect soluble Fas*Ex3/4/6. One (clone G1.6) reacted with an amino acid sequence encoded by Fas exon 2 and the other (clone B8.8) with non0-Fas C-terminal of this molecule. Some sera from active SLE patients were found to contain Fas*Ex3/4/6 by a newly-designed sandwich ELISA using these mAbs.
4. Preliminary experiments in the production of soluble Fas*Ex6 by human T cell lines such as Jurkat cells which were transfected with this gene did not work. Thus result suggested that an unknown regulatory mechanism(s) in the secretion of soluble FAS existed in T cell lines.
|
