2005 Fiscal Year Final Research Report Summary
Control of pulmonary fibrosis by the intracellular transfection of Smad protein
| Project/Area Number |
16590765
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Respiratory organ internal medicine
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| Research Institution | Nippon Medical School |
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Principal Investigator |
USUKI Jiro Nippon Medical School, Faculty of Medicine, Assistant Professor, 医学部, 講師 (80318477)
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| Co-Investigator(Kenkyū-buntansha) |
AZUMA Arata Nippon Medical School, Faculty of Medicine, Associate Professor, 医学部, 助教授 (10184194)
AOYAMA Akinori Nippon Medical School, Faculty of Medicine, Assistant Professor, 医学部, 講師 (60089688)
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| Project Period (FY) |
2004 – 2005
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| Keywords | TGF-β / Smad / fibroblast / collagen / α-smooth muscle actin / protein transfection / pulmonary fibrosis |
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| Research Abstract |
Smad proteins are known as the main signal pathway of TGF-β. In this study we investigated the effects of Smad proteins transfection to fibroblasts and the possibility of its clinical application for the treatment of pulmonary fibrosis. At first, using a mouse lungs fibroblast cell line (MLg) stimulated by TGF-β1, we analyzed the expressions of Smad proteins, type I collagen, and α-smooth muscle actin (ASMA).
As we found the induction of type I collagen through the activation of Smad pathway, type I collagen was suitable as a target molecule to evaluate the function of fibroblasts transfected by Smad proteins. The expression level of ASMA did not vary with TGF-β1 stimulation, suggesting the constitutional ASMA expression in this cell line. However, morphologically ASMA positive filaments were present within 1 hour after TGF-β 1 stimulation, and cell shape showed a change to myofibroblast. Myofibroblast might be induced by TGF-β without a change of ASMA expression level.
For human fetal lung fibroblast stock (HFL-1), we experimented on transfection with human Smad proteins. We used Chariot^<TM> as a carrier and reviewed the efficiency of protein transfection into cells by β-galactosidase which was a positive control. Under this condition, recombinant human Smad2 protein was transfected to fibroblasts. We found a significant increase of Smad2 protein in extract from transfected fibroblasts, but the change was absent in phosphorylated Smad2 and type I collagen. We showed that we could easily introduce Smad proteins into fibroblasts with this method.
We are going to study whether transfected Smad proteins can function in a cell in future.
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