Study On Effects And Mechanisms Of IFN-γ On TGF-β/Smad Signal Pathway In Pathological Scar Tissues Derived Fibroblasts

【Background】Pathological scars include hypertrophic scars and keloids. Their common feature is a collagen-based extracellular matrix (ECM) metabolism disorder, resulting in a large number of abnormal extracellular matrix deposition, which leads to the deformities and dysfunction of the patients. Studies show that TGF-βis the key cytokine that leads to scar formation. It regulates proliferation, apoptosis, trans-differentiation and collagen metabolism of the fibroblasts mainly through the TGF-β/Smad signaling pathway. Interferon-γ, a medicine belonging to cytokine family, can exert anti-virus, anti-tumor and immunomodulatory effects. In addition, some studies show that it can be used for the treatment of fibrotic diseases such as liver fibrosis, pulmonary fibrosis, hypertrophic scar and systemic sclerosis. However, the specific mechanism by which interferon-γexerts anti-fibrosis effects, especially its effects on pathological scar tissues derived fibroblasts TGF-β/Smad signaling pathway and the mechanism remains unclear. 【Purposes】1. To observe the effects of IFN-γon the expression level ofα-SMA, MMP1, TIMP1 and CTGF induced by TGF-β1 in pathological scar tissues derived fibroblasts, and to elucidate mechanisms of anti-fibrosis by IFN-γ.2. To find out the effects of the IFN-γon the expression level of Smad3 and Smad7 in pathological scar tissues derived fibroblasts at different time and concentration, and to investigate the mechanism preliminarily.【Contents】1. Effects of IFN-γon the expression level of CTGF: real-time quantitative PCR and Western blot were used to detect the expression level of CTGF gene and protein.2. Effects of IFN-γon the expression level ofα-SMA, MMP1 and TIMP1 induced by TGF-β1: Cytochemical immunofluorescence and Western blot were used to detect the expression ofα-SMA, and the real-time quantitative PCR was used to detect the expression level of MMP1 and TIMP1.3. Effects of IFN-γon the expression level of Smad3 and Smad7 at different time and concentration in fibroblasts: real-time quantitative PCR and Western blot were used to detect the expression level of Smad3 and Smad7 gene and protein.4. Construction of YB-1 over-expression vector; and YB-1 shRNA vector.【Results】1. IFN-γcan suppress the expression level of CTGF when used alone. Besides, when used with TGF-β1, it had a strong antagonistic effect on the mRNA level of CTGF induced by TGF-β1.2. IFN-γcan reduce the expression ofα-SMA in basic state or induced by TGF-β1 in the hypertrophic scar tissues derived fibroblasts. When used alone, IFN-γhad no significant effects on the expression level of MMP1 and TIMP1. However, it can inhibit the effects of TGF-β1 on MMP1 and TIMP1 when used with TGF-β1.3. IFN-γcan down-regulate the expression of Smad3 and increase the expression of Smad7 in a time- and dose-dependent manner, by inducing or inhibiting gene transcription.4. YB-1 over-expression of YB-1 vectors and the shRNA vectors were constructed successfully.【Conclusion】1. IFN-γcan inhibit the expression of CTGF induced by TGF-β1, which may be a mechanism of importance in the management of pathologic scar by IFN-γ.2. IFN-γcan significantly reduce the expression ofα-SMA in the basic state or induced by TGF-β1 in the scar-derived fibroblasts.3. IFN-γcan down-regulate the expression level of Smad3 while up-regulate the expression level of Smad7, which exerted the antagonistic effects on the TGF-β/Smad signal pathway.