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(PP210) Inhibiting the effects of TGF-1 on increasing collagen formation in fibroblast: the application of replication-defective adenovirus containing radio-inducible Egr-1 promoter and smad7 cDNA under ionizing radiation.
Cai, Xu Wei*,1, Fu, Xiao Long1, 1 Fudan University Cancer Hospital, Shanghai, China
ABSTRACT- Purpose/Objective: To evaluate whether replication-defective adenovirus recombinated with Egr-1 promoter and Smad7 cDNA can express Smad7 protein in fibroblast cells under ionizing radiation, and further block the signal transduction of transforming growth factor- 1 (TGF-1) in vitro. Materials/Methods: Based on the Adeno-X expression system, the CMV promoter of the pShuttle vector was replaced by Egr-1 promoter, and the Smad7 cDNA was subcloned into the MCS(multiple cloning site) of pShuttle. The recombinant pShuttle was then subcloned into the Adeno-X genome, which was transformed into E.coli to get recombinant Adeno-X plasmid DNA. The recombinant adenovirus was made and amplified in the transfected HEK 293 cells before it was purified and tested for viral titer. The fibroblast cells (3T6 cells) infected by the recombinant adenovirus were irradiated, and the activity of Egr-1 promoter was quantitively determined by the amount of Smad7 protein expressed in the 3T6 cells using Western blot. In addition the activity of preventing radiation-induced fibrosis by Smad7 protein was tested by the amount of collagen synthesization and proliferation of 3T6 cells with the technique of 3H-Proline incorporation and 3H-Thymidine incorporation. Results: Identified by restriction endonuclease analysis and PCR, recombinant adenovirus with Egr-1 promoter and Smad7 cDNA was constructed successfully, with a viral titer of 1.0x 1011 TCID50/ml. The expression level of Smad7 protein varied at different dose levels and different post-irradiation time points in the 3T6 cells infected with recombinant adenovirus. The amount of Smad7 protein increased in proportion to the rise in dose of radiation, and remained at a high expression level from 8 Gy to 15 Gy. The amount of Smad7 protein started to increase at 2 hours after irradiation, and remained steady for the next 5 hours before it descended, which was not observed in the control 3T6 cells.When 3T6 cells were cultured with TGF-1, the cells infected with the recombinant adenovirus synthesized significantly less collagen after irradiation than the cells without infection (P 0.001).(Figure 1) However, there were no differences in the proliferation of infected or uninfected 3T6 cells (P 0.312). Conclusions: With the aid of Adeno-X expression system and molecular cloning techniques, construction of recombinant adenovirus can be quick and efficient. The increase in Smad7 expression by control of Egr-1 promoter under ionizing radiation was time- and dose-dependent as determined by Western blot analysis. The signal transduction of TGF-1 can be blocked by the expressed Smad7 protein in 3T6 cells. As the next step, the prevention of radiation-induced lung fibrosis treated by the recombinant adenovirus in mouse will be studied in our laboratory. This work is supported by National Nature Science Foundation of China (30170289).
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