The In Vivo Study On The Effect Of Adenovirus Mediate Smad7 Gene Expression Regulated By Irradiation Via Egr-1 Promoter To Prevent Radiation-induced Lung Fibrosis

Radiation-induced lung fibrosis is a common complication in patients received the radiotherapy for their thoracic tumour. Radiation-induced lung fibrosis could influences the quality of life of patients and even lead some patients to death. Owing to the mechanisms of radiation-induced lung fibrosis is not very clear by now, there are only symptomatic treatment and valid therapeutics are insufficient. Respecting the further research of the mechanisms of radiation-induced lung fibrosis and the rapid development in molecular biology, gene therapy is becoming a significant research area and probably to be a new significant therapeutics. Recent investigations demonstrate that radiation-induced lung fibrosis is the consequences of the inflammatory reactions between multiple cytes and systems with multiple cytokines participating and the transforming growth factor-beta (TGF- β ) is thought as one of the most important cytokines. Smad7 is a inhibitor of the cellule signal transmission of transforming growth factor-beta in cytoplasm. Studies already illustrate that bleomycin-induced lung fibrosis could be prevent by exogenous Smad7 gene to blocking the cellule signal transmission of transforming growth factor-beta. In account of the generally existence and effectiveness of transforming growth factor-beta in organism, it probably cause a series of harmful effects to organism when blocking the cellule signal transmission of transforming growth factor-beta diffusely. Accordingly, it is criticality to blocking the cellule signal transmission of transforming growth factor-beta regional and it is the key point to determine whether the therapeutic device to prevent radiation-induced lung fibrosis is available in clinical.Early growth response-1 is a radiosensitive promoter, it could active the following gene expression selectively in the radiation field. If we could utilize the radiation-inducible promotor to control Smad7 gene transcription, the Smad7 proteincould be expressed to block transforming growth factor-beta signal transmission only in specific irradiate region, maybe we could prevent Rdiation-induced lung fibrosis targetingly. But there is no analogical research by now.So the replication defective adenovirus wich was recombinated with early growth response-1 promoter and Smad7 cDNA was constructed in our laboratory. We use C57BL/6j mice to construct the radiation-induced lung fibrosis model to study the mechanisms and validity of the recombinated adenovirus to block transforming growth factor-beta signal transmission and prevent radiation-induced lung fibrosis targetingly. We also study the contribution of the recombinated adenovirus to mice implanted with lewis lung cancer and to demonstrate the safety of recombinated adenovirus to prevent radiation-induced lung fibrosis. We do the translation research and it may present a new therapeutics theory for radiation-induced lung fibrosis in clinical.Objective To investigate the safe concentration of AD.Egr-Smad7 for C57BL mouse and the orientation of the expression of exogenous Smad7. To demonstrate the time-effect relationship and dose-effect relationship of the expression of adenovirus mediating Smad7 gene regulated by irradiation via early growth response-1 promoter in the lungs of C57BL mouse. To illustrate the effects and the mechanisms of adenovirus mediate Smad7 gene expression regulated by irradiation via early growth response-1 promoter to prevent radiation-induced lung fibrosis in C57BL/6j mice. To study the contribution of adenovirus mediate Smad7 gene regulated by irradiation via early growth response-1 on the primary tumor growth and lung metastasis in C57BL mice implanted with Lewis lung cancer.Methods The recombinant adenovirus (AD.Egr-Smad7) made up of replication-defective adenovirus enclosed radio-inducible elements from the early growth response-1 gene promoter and cDNA encoding Smad7. Mice were randomly divided into groups, then they were infected by intratracheal instillation with AD.Egr-Smad7 and irradiated to their whole lungs after 24 hours. They were infectedby intratracheal instillation with different concentration AD.Egr-Smad7 or adenovirus vector respectively to study the toxicity of the virus with different concentration compared with the saline group. Immunohistochemical method was used to to study the orientation of the expression of extrinsic Smad7. Mice were infected by intratracheal instillation with different concentration AD.Egr-Smad7 , the lungs were harvested at different time point following irradiation and mice were irradiated respectively by different single irradiation dose to observe the time-effect relationship, dose-effect relationship of exogenous Smad7 gene expression. The expression of exogenous Smad7 was detected by Western blotting analysis. I and III collagen synthesized , the transforming growth factor beta(TGF β) and the connective tissue growth factor(CTGF) in lung tissue were detected by ELISA analysis. Hydroxyproline in lung tissue was detected by Alkaline Hydrolyze Specimen. Hematoxylin and eosin staining were performed and the degree of lung fibrosis was graded. All of the above were detected at different time interval after irradiation to investigate the effects and the mechanisms of adenovirus mediate Smad7 gene expression regulated by irradiation via early growth response-1 promoter to prevent radiation-induced lung fibrosis in C57BL/6j mice. To record the maximal and minimal diameters of the tumor to observe the tumor growth tendency, the tumor growth delay and the mice survival time; to observe the frequency of lung metastases after two weeks of radiation; to observe the frequency of lung metastases when the tumor volume was four times as big as that of the radiation initiation in order to study the contribution of adenovirus mediate Smad7 gene regulated by irradiation via early growth response-1 on the primary tumor growth and lung metastasis in C57BL mice implanted with Lewis lung cancer.Results 1. The expression of adenovirus mediating Smad7 gene which is regulated by early growth response-1 gene promoter could be induced by irradiation markedly compared with control groups (OR=0.2, P=0.0000, P<0.01). It is safety for mouse when the concentration of AD.Egr-Smad7 is 10~9 pfu and below. There is expression of exogenous Smad7 in cytoplasm of bronchial epithelium cells, the alveolar ductsand alveoli cells and alveolar wall blood capillaries cells. 2. The degree of Smad7 gene expression has relationship with adenovirus dose, irradiation dose and time interval after irradiation. The expression degree of Smad7 gene increased in a dose-dependent manner when the AD.Egr-Smad7 dose was at 10~8,5 ×10~8,10~9 pfu (P <0.01)and the expression degree of Smad7 gene also increased in a dose-dependent manner when the irradiation dose was at 0-12Gy, then it decreased when the irradiation dose was at 15Gy. The expression degree of Smad7 gene appeared to reach a peak at 2-9 hours after initiation of irradiation (P<0.01) and then regulated down and almost returned to the normal standard 15 hours later. 3. After irradiation, AD.Egr-Smad7 could reduce the content of I and IIIcollagen synthesized , TGF β , CTGF and Hydroxyproline in lung tissue markedly compared with control groups (P < 0.05). The histopathologic alterations of radiation-induced lung fibrosis in AD.Egr-Smad7 induced by γ -ray irradiation group is lightened compared with control groups. 4. The adenovirus mediate Smad7 gene expression regulated by irradiation via early growth response-1 gene promoter in C57BL mice implanted with Lewis lung cancer could inhibit the progression of the primary tumor and prolong the tumor growth delay and the mice survival time significantly compared with the control group (tumor growth delay RR=0. 488, P=0. 0214, P<0. 05; mice survival time RR=0. 530, P=0. 044, P<0. 05), but it could not significantly inhibit the tumor lung metastases (two weeks after radiation initiation AD.Egr-Smad7 group compaired with blank group P=0. 1300, P>0. 05; blank group compaired with NS group P=0.7203, P>0. 05; when the tumor volume was four times as big as that of the radiation initiation AD.Egr-Smad7 group compaired with blank group P=0. 5610, P > 0. 05 ; blank group compaired with NS group P=1, P >0. 05 ) . Conclusion 1. The in vivo investigation demonstrates that γ -ray irradiation markedly induce adenovirus mediating Smad7 gene expression regulated by early growth response-1 gene promoter. It is safety for mouse when the concentration of AD.Egr-Smad7 is 10~9 pfu and below. There is expression of exogenous Smad7 in cytoplasm of bronchial epithelium cells, the alveolar ducts and alveoli cells and alveolar wall blood capillaries cells. There is the possibility of AD.Egr-Smad7 toblock the signal transduction of TGF- β of C57BL mouse lung in tolerance and orientation aspect. 2. The in vivo investigation demonstrates that γ irradiation markedly induce adenovirus mediating Smad7 gene expression regulated by early growth response-1 gene promoter. The expression of Smad7 gene has correlation with AD.Egr-Smad7 dose, irradiation dose and time interval after irradiation. This regularity demonstrate that the expression of exogenous Smad7 to blocking the signal transduction of TGF- β locally as a novel strategy for gene therapy aiming at preventing radiation-induced lung fibrosis deserves further study. 3. The in vivo investigation demonstrates that AD.Egr-Smad7 to block the signal transduction of TGF- β locally as a novel strategy for gene therapy aiming at preventing radiation-induced lung fibrosis can prevent radiation-induced lung fibrosis in C57BL/6j mice to some extent. 4. The gene expression of AD.Egr-Smad7 regulated by irradiation has no risk in promoting local progression and distant metastasis of Lewis lung cancer in mice. On the other hands, the gene expression of AD.Egr-Smad7 regulated by irradiation could inhibit the progression of the primary tumor and prolong the tumor growth delay and the mice survival time significantly. It is safe to some extent of using AD.Egr-Smad7 to blocking the signal transduction of TGF- β locally as a novel strategy for gene therapy aim at preventing radiation-induced lung fibrosis. 5. Our translation research present a new therapeutics theory for preventing radiation-induced lung fibrosis targetingly in clinical and it deserves further study.