| Budget Amount *help |
¥14,800,000 (Direct Cost: ¥14,800,000)
Fiscal Year 2004: ¥6,800,000 (Direct Cost: ¥6,800,000)
Fiscal Year 2003: ¥8,000,000 (Direct Cost: ¥8,000,000)
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| Research Abstract |
Analysis of in vitro cell functions with β3 cDNA-transduced human keratinocytes
Human keratinocytes transduced with β3 integrin subunit cDNA by a retrovirus-mediated transduction method expressed β3 and αvβ3 on the cell surface. The percentage of positive β3 expressed cells with the cultures of β3 cDNA-transduced keratinocytes having 20-30%, 50-60%, 70-80% and 100% confluence were 69%, 68%, 65% and 33%, respectively. On the other hand, the control β-gal cDNA-transduced keratinocytes were negative for β3 or αvβ3. The transduction of β3 integrin subunit cDNA did not affect the expression of other αv integrin complexes such as αvβ5 or αvβ6. One hour cell adhesion assays demonstrated that the β3 cDNA-transduced human keratinocytes adhered to fibrin (FB), fibrinogen (FG), vitronectin (VN), fibronectin (FN) and denatured collagen { gelatin (GEL) } significantly compared with β-galactosidase (β-gal) cDNA-transduced keratinocytes (control). Inhibition assay of the cell adhesion to FB and GEL wa
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s performed using cells which were incubated with various concentrations of monoclonal antibody to αvβ3 (LM609) or normal mouse IgG1 or alternatively with peptides GRGDSP or GRGESP for 30 min at RT before adding cells to the wells. The adhesion of the β3 cDNA-transduced human keratinocytes to FB and GEL was inhibited by the LM609 and RGD peptides in a dose-dependent fashion, but not by the normal mouse IgG1 nor RGE peptides. Five days cell growth assays showed that β3 integrin subunit cDNA-transduced keratinocytes grew on FB, FG, VN and denatured collagen (GEL) significantly more than the β-gal cDNA-transduced keratinocytes (control). The cell growth rate, calculated by the increased rate of cell numbers from one day to five days, of the β3 cDNA-transduced keratinocytes also increased on FG, FB, VN and GEL significantly more than the control. There were no differences in the cell growth on laminin, type IV collagen and type I collagen between the two groups. Furthermore, 14 hours haptotaxis migration assays revealed that the β3 cDNA-transduced keratinocytes migrated to FG, FB, GEL, VN and FN significantly more than the control cells did. Both groups of cells migrated to type I collagen to the same degree and did not migrate to BSA. A monoclonal antibody to αvβ3 (LM609) considerably inhibited the migration of the β3 cDNA-transduced keratinocytes to FG, FB, GEL, VN and FN.
Thus, these data support the idea that these recombinant keratinocytes provide a method of enhancing wound healing in a graft procedure when they are applied to FB, FG and denatued collagen-rich cutanous wounds. Less
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