| Co-Investigator(Kenkyū-buntansha) |
TANAKA Shinya Hokkaido Univ., Grad. School of Med., Inst., 医学部, 助手 (70261287)
SAWA Hirofumi Hokkaido Univ., Grad. School of Med., Asso. Pro., 医学部, 助教授 (30292006)
鈴木 宏明 北海道大学, 医学部, 助手 (40281802)
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| Budget Amount *help |
¥10,200,000 (Direct Cost: ¥10,200,000)
Fiscal Year 1999: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1998: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 1997: ¥7,800,000 (Direct Cost: ¥7,800,000)
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| Research Abstract |
We have established a rat model of injury of cerebellar granule cells by intoxication of methylmercury chloride (MMC). To elucidate the molecular mechanisms of brain damage induced by MMC we initial ly examined the pathologic examination by H&E staining, electron microscope, TUNEL staining, and DNA fragmentation. These examination revealed that cerebellar granule cell death by organic mercury intoxication occurs via an apoptotic process and was restricted to the granule cells and that the distributional pattern of apoptotic cerebellar granule cells was specific in rat cerebellar vermis. Secondly, we investigated expression levels of apoptosis related molecules such as; BDNF, Trk B, Fas ligand, RIP, Akt, Bad, Bcl-2, Caspase 9, Caspase 3, CAS, and TIAR using Western blot and also evaluated the function of Purkinje cells by immunostaining with anti-calbindin D. Moreover, we investigated the alteration of gene expression of rat brain after MMC intoxication. These results demonstrated that
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expression of BDNF and Trk B were up-regulated, while RIP, CAS, Fas ligand, Bad, and Caspase 9 were down-regulated. The findings suggest that the up-regulated molecules function to suppress apoptosis at this stage. Gene expression analysis disclosed that among up-regulated genes 1) those related to apoptosis were protein kinase C γ, interleukin 13, insulin receptor substrate Z, cyclophilin, protein phosphatase 1, and RL/IF-1, 2) those related to hormone were vasopressin and oxytocin, 3) those related to enzyme were serum and glucocorticoid-regulated kinase, mast cell protease, serine protease, glycoprotein specific UDP-glucuronyl transferase, protein tyrosine phosphatase, 4) 7 miscellaneous molecules, and 5) 7 unknown genes. In contrast, down-regulated genes were 2α1 globin gene and 2 unknown genes. The results could be summarized that apoptosis suppressive genes were activated at the beginning of cerebellar apoptosis. Thus, it could be concluded that in the initial stage of MMC intoxication the expression of anti-apoptotic molecules were more enhanced to protect cell death than apoptosis-inducing molecules. Less
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