2000 Fiscal Year Final Research Report Summary
Development of artificial endocrine organs composed of cultured skin cells
| Project/Area Number |
09557096
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| Research Category |
Grant-in-Aid for Scientific Research (B).
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
General surgery
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| Research Institution | Tokai University |
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Principal Investigator |
INOKUCHI Sadaki Tokai University, School of Medicine, Associate Professor, 医学部, 助教授 (60160008)
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| Co-Investigator(Kenkyū-buntansha) |
ANDO Kiyoshi Tokai University, School of Medicine, Assistant Professor, 医学部, 講師 (70176014)
SHIMAMURA Kazuo Tokai University, School of Medicine, Associate Professor, 医学部, 助教授 (00119679)
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| Project Period (FY) |
1997 – 2000
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| Keywords | virus vector / keratinocytes / diabetes / insulin / GFP / lentiviral vector |
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| Research Abstract |
A purpose of this study was to develop artificial endocrine organs by introducing genes of hormones or cytokines into human epidermal keratinocytes.
(1) To increase infectivity of a retrovirus vectorwe constructed an adenovirus vector into which gene of an amphotropic retrovirus receptor is cloned. Although infectivity of a retrovirus vector to K562 cells were increased, infectivity to human epidermal keratinocytes was not changed after infection of the adenovirus.
(2) We constructed a retrovirus vector into which both enhanced green fluorescent protein (GFP) gene and human preproinsulin cDNA were cloned. After infection of the retrovirus vector, GFP positive human epidermal keratinocytes were selected by FACS sorting. The procedure enriched GFP positive keratinocytes to10 times. These GFP positive cells stably produced proinsulin for 14 days in vitro. Although, when the cells were transplanted into immunodeficient mice, expression of both GFP and proinsulin were despaired within 4 weeks
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(3) GFP and preproinsulin genes were cloned into MSCV retrovirus vector, which had altered LTR sequence. To measure the stability of introduced genes in vivo, human keratinocytes were infected by the vector and transplanted into immunodeficient mice. However, expression of the introduced genes could not be detected at 4 weeks after transplantation
(4) A replication-defective vesicular stomatitis virus glycoprotein G pseudotyped HIV-1-based vector encoding the enhanced GFP was used for gene transfer into human keratinocytes. Afier infection of the lentiviral vector, 10% of the cells showed GFP positive.
These results showed that enrichment of gene introduced cells using GFP marker and FACS sorting was a useful procedure for human keratinocytes. However, two major problems of conventional recombinant retrovirus vectors, low infectivity and unstable expression of introduced gene in vivo, were still remained. We considered that HIV-1 based lentiviral vector was a promising alternative for efficient and stable gene transfer into human epidermal keratinocytes. Less
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