1993 Fiscal Year Final Research Report Summary
Mechanism of new regeneration activating factor of aged nervous tissue secreted from hepatocytes and its determination with vitro immunization
| Project/Area Number |
04836020
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
老化(加齢)
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| Research Institution | Yokohama City University |
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Principal Investigator |
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| Project Period (FY) |
1992 – 1993
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| Keywords | tissue culture / collagen gel / neural regeneration / adult murine / hepatocyte / enhancement factor / retina / dorsal root ganglion |
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| Research Abstract |
We have established culture system of central and peripheral nervous tissues of matured or aged murine by introduction of three-dimensional collagen gel culture. Retina was used as a central nervous tissue and dorsal root ganglion was as a peripheral one. Hepatocyte secreted factor was applied to these cultures to demonstrate its activity, mechanism, and determination of the factor enhancing neural regeneration. Hepatocytes were dissected from adult rats by collagenase perfusion method and cultured in a serum-free William's E medium. The culture medium was applied to cultured retina explants as hepatocyte conditioned medium(HCM). Retina was removed from an adult rat 7 days after optic nerve transection and dissected to small fragment. They were embedded in collagen gel and cultured in the same medium as that in hepatocytes. Processes appeared 24 h after in culture. Immunocytochemical staining revealed that these processes were positive either to anti-neurofilament antibody or anti-thy-1.2 antibody. These results clarified that regenerating processes were neurites from retinal ganglion cells. When HCM was applied to a retina explant, the number of regenerating neurites increased to 2.6 times larger than that in control medium. This enhancement activity appeared in 5k< <100k fraction after membrane filtration and disappeared after the treatment with 95ﾟC, 5 min. This factor enhanced neurite regeneration from transected nerve terminals of adult or aged murine dorsal root ganglion (DRG), too. Since the explants of retina or DRG kept structures and functions similar to the tissues in situ, this factor is expected to work in vivo as a neural regeneration activating factor. We have already examined 20 substances recognized as a growth factor or a neural regeneration activating one, but no one exerted the same effect as a hepatocyte secreted one. This factor is thought to be new. We have developed purification of the substance and it will reach a final stage to decide the
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