| Co-Investigator(Kenkyū-buntansha) |
KASAI Sin-ichi Asahikawa Medical College, Professor, 医学部, 教授 (40091566)
ONODERA Kazuhiko Asahikawa Medical College, Associate Professor, 医学部, 講師 (00204264)
松田 年 旭川医科大学, 医学部, 助手 (90312470)
MATSUDA Minoru Asahikawa Medical College, Associate (50235157)
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| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 1999: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1998: ¥2,900,000 (Direct Cost: ¥2,900,000)
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| Research Abstract |
We investigated the long-term effect of transgenic expression of HGF/SF on survival and proliferation of transplanted hepatocytes in the mouse spleen using HGF/SF transgenic mice. In contrast to intrasplenic hepatocyte transplantation in rat, wild type hepatocytes isolated from FVB/N mice did not show any survival in the syngeneic mouse spleen. Whereas, transgenic hepatocytes grew and survived in the syngeneic mice spleen with creating significant cell clusters. The hepatocytes number in the longitudinal section of the spleen was quantitated by NIH/Image software. Although the number of transgenic hepatocytes in the spleen gradually decreased until 16 weeks after transplantation, it started to increase at 32 weeks after transplantation. At 64 weeks after transplantation, the number of transplanted hepatocytes nearly reached to the 5 % of host liver volume. Further, in every time points we observed from 2 to 64 weeks, the number of transgenic hepatocytes in the spleen is superior than t
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hat of wild type hepatocytes with a statistically difference. In the high power magnification, we observed hepatocytes cluster which showed quite similar with sinusoid structure in the liver at 64 weeks, suggesting that transplanted hepatocytes may reconstitute the liver architecture with well differentiated phenotype of hepatocytes in the spleen. To demonstrate the function of transplanted hepatocytes in the spleen, we examined the expression of transcripts and protein of various hepatic markers, such as transgene HGF/SF, albumin, cytokeratin 18 and glycogen, determined by PAS staining. As a result, transgenic hepatocytes in the spleen expressed the transcripts of transgene HGF/SF, albumin and cytokeratin 18 as a time dependent manner from 4 to 32 weeks after transplantation. At 64 weeks after transplantation, transplanted hepatocytes still produce albumin and glycogen demonstrated by immuno-histochemistry and PAS staining, respectively. To determine the mitogenic effect of HGF/SF on transplanted hepatocytes, we examined the DNA synthesis of transplanted hepatocytes by PCNA staining. Wild type hepatocytes showed negative staining of PCNA. In contrast, transgenic hepatocytes exhibit positive staining of PCNA. The frequency of DNA synthesis reached a peak at 8 weeks after transplantation and decreased gradually until 64 weeks. Taken together, over-expression of HGF/SF in hepatocytes as an autocrine manner enhanced the survival and the proliferation of transplanted hepatocytes in the spleen for a long term period. Furthermore it also sustained the hepatocyte function of transplanted hepatocytes in the spleen. Currently we are trying to investigate the effect of transgenic hepatocytes transplantation in mouse disease model which is deficient of certain enzyme specific for hepatocytes. Specifically we will transplant the tarnsgenic hepatocytes into the spleen of mdr2 knockout mouse which is thought to be a disease model of progressive fainiliar intrahepatic cholestasis (PFIC). Less
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