Normal human astrocytes (NHAs) in vitro grow slowly at a rate of about 7-10 days for 1 population doubling. ' They showed a limited proliferative lifespan at a maximum of 20 population doubling level (PDL) showing phenotypes of senescent cell such as large and flattened morphology and senescence-associated β-gal expression without apparent telomere shortening. We established lifespan-extended NHAs after introduction of telomerase reverse transcriptase gene (hTERT). hTERT-introduced NHAs expressed telomerase activity, maintained telomere size, and proliferated continuously over 100 PDL with normal phenotypes such as growth rate, cell morphology, karyotype, and GFAP (glial fibrillary acidic protein) expression. Senesced normal NHAs at 19 PDL down-regulated the expression of GFAP and upregulated the expressions of PDGF-β, FGF-2, GM-CSF, TGF-α, s100-β, IL-6, NT-4, NGF-β, and M-CSF as compared with those at 10 PDL. Expression level of these genes was recovered in hTERT-introduced to the level of NHAs at 10 PDL. Expression levels of FGF-1, EGF, IFN-α, CNTF, and NT-3 did not change markedly by senescence or hTERT introduction. These results suggest that NHAs maintained not only proliferative potential but also cellular functions such as cytokine expression by the introduction of hTERT. Proteome analysis revealed overexpression of anti-apoptotic proteins (clusterin, fortilin, and gelsolin) and down-regulation of apoptotic protein (calreticulin) in hTERT-introduced NHAs, although the role of these proteins on lifespan extension was unknown.

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Subject to the ethics guideline for human genome/gene analysis research enforced in Japan, authorization for the research protocol was obtained from the institutional ethics review committees. Peripheral blood samples were then collected after providing adequate explanation from Japanese volunteers, and obtaining written consent based on free will. Prior to use, the collected blood samples were anonymized in an unlinkable fashion on the new program developed by us, and B-cell lines derived from 996 individuals were established by the exposure of their blood cells to Epstein-Barr virus. All the established cell lines were deposited to the public cell banks, National Institute of Health Science and Japan Health Science Foundation, and their distribution was begun in 2003. The distribution of these cell lines available as materials for human gene analysis research, may contribute to the advancement of human gene analysis research.

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A drug-hypersensitive PC12 mutant cell line (PC12m3) was obtained during continuous culturing of neural PC12 cells. In this study, PC12m3 cells were exposed to vibratory sound stimuli of frequencies ranging from 10 to 200 Hz for 30 minutes at intensity 5 under the condition of NGF treatment. The results showed that low-frequency vibratory sounds of 10-100 Hz induced enhancement of neurite outgrowth, whereas vibratory sounds 150 Hz and 200 Hz had little effect on neurite outgrowth. The frequency of neurite outgrowth induced by 40-Hz low-frequency vibratory sound stimuli was approximately 3-fold greater than that induced by NGF alone. The activation of p38 MAPK has been shown to play an important role in neuronal differentiation in PC12m3 cells. Therefore, we examined whether the ability of low-frequency vibratory sound stimulus to induce neurite outgrowth of PC12m3 cells is a reflection of its effect on p38 MAPK activity. The results demonstrated that 40-Hz low-frequency vibratory sound stimuli had an enhancing p38 MAPK activity and indicated that vibratory sound induces neurite outgrowth via a p38 MAPK signaling pathway in PC12m3 cells.

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[in Japanese]

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