Objective: To investigate the regular pattern of expression, and expression localization in lung of Smad7 gene mediated by adenovirus and regulated by irradiation via early growth response (Egr) factor-1 promoter and the safe dose of the recombinant adenovirus.
Methods: The radio-inducible element of Egr-1 gene promoter and cDNA encoding Smad7 were enclosed into the replication-defective adenovirus, thus establishing the recombinant adenovirus AD.Egr-Smad7. 720 C57BL/6J mice were randomly divided into 6 different groups: Group C (blank control group, receiving normal saline only), Group R (receiving radiation with the dose of 8 Gy at the whole chest only), Group AR (undergoing intratracheal instillation of AD.Egr-Smad7 without radiation), Group RAR (undergoing intratracheal instillation of AD.Egr-Smad7 and then radiation 24 hours later), Group AV (receiving the replication-defective adenovirus without radiation), and Group AVR (receiving both the replication-defective adenovirus and radiation). The following studies were conducted. (1) Tolerance to the adenovirus. Sixty mice were divided into 11 equal subgroups: Subgroups C, and Subgroups RA and AV receiving AD.Egr-Smad7 at 5 different concentrations: 10(8), 5 x 10(8), 10(9), 5 x 10(9), and 10(10) pfu/0.1 ml respectively. The mice were observed for the symptoms of dyspnea and cyanosis and death within 72 hours. (2) Localization of the expression of exogenous Smad7 in the lungs. 36 mice were divided into 6 equal Subgroups: C, R, RA, RAR, AV, and AVR, the adenovirus titre being 10(9) pfu/0.1 ml. Five hours after the irradiation all mice were killed to take out their lungs. (3) Relationship between the titre of AD.Egr-Smad7 and expression of Smad7 in lung. 168 mice were divided into 28 Subgroups: C, R, RA, RAR, AV, and AVR, the titre of virus being 10(8), 5 x 10(8), or 10(9) respectively, and the time after irradiation being 6 hours or 9 hours. The mice were killed at specific time points and their lungs were taken out. (4) Time-effect on Smad7 expression in lung. 134 mice were divided into 54 equal Subgroups: R, RA, RAR, AV, and AVR. The mice were killed 0, 1, 2, 3, 5, 7, 9, 12, and 15 hours after irradiation and their lungs were taken out. (5) Dose-effect of irradiation on the Smad7 expression in lung. 126 mice were divided into 21 Subgroups: C, R, RA, RAR, AV, and AVR, the irradiation dose being 5, 8, 10, 12, 15, or 20 Gy. Five hours after the irradiation the mice were killed and their lungs were taken out. Western blotting was used to detect the mRNA expression of Smad7 in lung and the protein expression of Smad7 in lung was examined by immunohistochemistry.
Results: (1) The concentration of 10(9) pfu and lower was safe, no matter the adenovirus contained Egf-Smad7 or not. (2) mRNA expression of exogenous SmaD7 was found in the cytoplasm of bronchial epithelium cells, alveolar epithelial cells, and blood vessel endothelial cells in Subgroup RAR but not in the other subgroups. (3) Western blotting showed that the protein expression of Smad7 was obvious in the lung tissues of Subgroup RAR, being more obvious 6 hours after irradiation than 9 hours later (P < 0.01), AD.Egr-Smad7 dose-dependently increased the protein expression of exogenous Smad7 in Subgroup RAR (P < 0.01), and that the protein expression of Smad7 was very weak in the other subgroups in comparison with Subgroup RAR (all P < 0.01). (4) The protein expression of Smad7 in the lung tissues of Subgroup RAR began to increase 1 hour after irradiation, peaked 5 hours later, then gradually decreased, returned to the level 1 hour later by the 12 th hour after irradiation, and almost disappeared 15 hours later. (5) The protein expression of Smad7 in Subgroup RAR increased after irradiation in a dose-dependent manner (P < 0.01), with a peak when the dose was 12 Gy.
Conclusion: Gamma irradiation markedly induces the expression of exogenous Smad7 gene mediated by recombinant adenovirus in the cells of lung tissues dose-dependently that is correlated with time interval as well. It is important to look for a safe titre of the recombinant adenovirus. Blocking the signal transduction of transforming growth factor-beta may become a novel strategy for gene therapy aiming at preventing irradiation-induced lung fibrosis.