1991 Fiscal Year Final Research Report Summary
treatment of diabetes mellitus by cultured skin transfected with insulin genes
| Project/Area Number |
01870034
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| Research Category |
Grant-in-Aid for Developmental Scientific Research
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| Allocation Type | Single-year Grants |
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| Research Field |
内科学一般
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| Research Institution | Hiroshima University (1990-1991)
Tokyo Metropolitan University (1989) |
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Principal Investigator |
YOSHIZATO Katutoshi Hiroshima University, Faculty of Science Professor, 理学部, 教授 (20095516)
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| Co-Investigator(Kenkyū-buntansha) |
YOSHIKAWA Etuo Kanebo Institute for Development, Head investigator, 開発研究所, 主任研究員
MUKAI Ryozaburo NIH Japan, Center of Primate, Head investigator, 霊長類センター, 主任研究官 (90133040)
YAMAZAKI Yoshimitu Osaka Univ. Medical School, Research Associate, 医学部, 助手 (40201834)
KAWAMORI Ryuzo Osaka University, Medical School Assistant Prof., 医学部, 講師 (00116021)
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| Project Period (FY) |
1989 – 1991
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| Keywords | Diabetes mellitus / Insulin genes / Artificial skin / Recombinant DNA / Collagen / Gelatin |
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| Research Abstract |
To develop techniques for transfecting insulin genes into cells of artificial skin, the present study has been performed, whose content was divided into the following three. Recombinant insulin genes were prepared by inserting cDNA of rat preproinsulin into vectors. Insulin was successfully expressed in NIH 3T3 cells and secreted from them when the recombinant DNA was transfected into them. We also succeeded in developing a gelatin-based porous sponge which is expected to be useful substrate for artificial skin transfected with insulin genes. The bio-compatibility of this sponge has been studied in vitraand in vivo. The results of the three studies are summarized as follows.
1. Preparation of recombinant insulin genes. cDNAs of rat preproinsulin were inserted into one of four vectors : pZipneoSV (X), pSV_2-his, pMAM-neo and pMAM-neo-s. Eive micrograms of three recombinants were transfected into 2.5xlO^5 cells of NIH 3T3 utilizing the method described by Chen and Okayama. The recombinant with pZip-neoSV (X) was successfully expressed in the cells and secreted at the concentration of 15uU/ml for 2 days. There was no difference in the rate of growth between nontransfected and transfected cells.
2. Development of a new type artificial skin. A new type of substrate was developed, which is a gelatin-based porous sponge overlaid with a thin layer of gelatin. Average pore size of the sponge Was 110 um. Sponges were given physical and physiological stability by introducing chemical cross-links.
3. Bio-compatibility test of the gelatin-based artificial skin. The artificial skin made of gelatin described above was subjected to bio-compatibility test. Sponges showed good affinity toward fibroblasts and epidermal cells of human origin, when the sponge was inoculated in vitro with the cells. Tissue reactions were found good when sponges were implanted into dermis up to 4 weeks.
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[Publications] Kono, K., Tanii, T, Furukawa, M., Mizuno, N., Kitajima, J., Ishii, M., Hamada, T., and Yoshizato, K.: "Cell Cycle analysis of human dermal fibroblasts cultured on or in hydrated type I collagen lattices." Arch. of Dermatol. Res.282. 1-5 (1989)
Description
「研究成果報告書概要(欧文)」より
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[Publications] Kono, K., Tanii, T, Furukawa, M., Mizuno, N., Kitajima, J., Ishii, M., Hamada, T., and Yoshizato, K.: "Parallel arrangement, growth inhibition and cell cycle phase analysis of human dermal fibroblasts cultured in collagen lattices." J. Dermatol.17. 2-10 (1990)
Description
「研究成果報告書概要(欧文)」より
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[Publications] Kono, K., Tanii, T, Furukawa, M., Nizuno, N., Kitajima, J., Ishii, M., Hamada, T., and Yoshizato, K.: "Correlation of contractility and proliferative potential with the extent of differentiation in mouse fibroblastic cell lines cultured in collagen lattices." J. Dermatol.17. 149-154 (1990)
Description
「研究成果報告書概要(欧文)」より
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