| Budget Amount *help |
¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1997: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1996: ¥1,000,000 (Direct Cost: ¥1,000,000)
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| Research Abstract |
Establishment of separation and culture method for septal cell in rat lung
It was known that, during the process of rat postnatal development, the number of septal cell increase to 5-7 day after birth and decrease after the these days. We digested to disperse these cell with collagenase and attempted to two separate method ; Centrifugal cell fractionation to cell suspension with Percoll density solution and 7% Nycodenz solution. We were able to gain septal cell rich fraction with both two method. Cell was cultured with 1O% fetal calf serum containing medium F-10 or F12.
The distributional change and relationship between caveola component, receptor to biologically active substance and culture time
1)The expression of alpha-smooth muscle actin was weak on day l and positive cekll was 20%, but, on day 3, became strong and positive cell arrive to 75% with immunofluorescence study. This tendency was also confirmed on immunoblotting. 2)Caveolin (cavela specific protein), IP3R (inositoll, 4,5-tr
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isphosphate receptor) and CaATPase (Ca pump) was similar distributed in cultured day 1 septal cell. But the distribution of caveolin became be different localization of IP3R and CaATPase in day 3 septal cell. 3)Using with antibodies to a peptide corresponding to the carboxyl terminus of human ETA and ETB (gifted by Dr.Takimoto, Ciba-Geigy Japan), immunofluorescent study showed the existence of ETA and ETB in septal cell.4) ET-1-Cy3 induced the construction of the cell and the internalized ET-1-Cy3 localized with dot-like structure in the cell. 5)These antibodies recognized many bands on Western blot, but in tunicamycin treated cell, antibodies recognized the correct bands for ETA and ETB.The pseudoband was concluded the results of glycosylation to ETR.In ultrathin cryosection and immunoelectron microscopy, ETA partly localized caveola in septal cells and ETB did caveola in endothelial cells
Vimentin-related antien and fodrin around lipid drolets in septal cells
1) Antibody to the antigen around lipid droplets (gifted by Dr.SM-Wang, Taiwan Natl.Univ.) was rim pattern around lipid droplets and filamentous structure in septal cells. 2) On immunoblot, this antibody recognized 190,160 and 57 kDa band. 3) This antibody recognized 57 kDa band as anti-vimentin antibody in the homogenate of rat eye ball lens. 4) Colcemid and Cytochalasin Binduced the disappearance of the rim Pattern. This was similar to that of fodrin. 5) These results showed that this antigen associate with actin, vimentin and fodrin localized around lipid droplets in septal cells. Less
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