Inhibiting Formation Of Scar-related Proteins By Blocking Two Sites Of TGF-β Signaling

Objective To investigate the efficiency of blocking two sites of TGF-β/Smads signaling by soluble transforming growth factor-β receptor Ⅱ (sTpRII) and dominant-negative mutant Smad4△M4, which has a deletion in the linker region, in inhibiting formation of scar-related proteins.Methods Two lentivirus vectors were established encoding sTβRII and Smad4AM4respectively. The cultured human fibroblasts (HFF-1) were infected with different concentrations of these two lentivirus vectors to determine the optimum multiplicity of infection (MOI). Then it was divided into6groups:co-infection group, sTβRII group, Smad4AM4group, negative virus group, positive control group and blank control group. HFF-1was infected with both of these two lentivirus vectors at a1:1ratio as co-infection group, and with one of the two vectors as sTβRII group and Smad4AM4group. HFF-1was infected with mock-vehicle lentivirus as negative virus group. The infected HFF-1s were then stimulated with TGF-β1. The positive control group was HFF-1simply stimulated with TGF-β1and the blank control group was HFF-1with no treatments. Western blot/qPCR, ELISA, and SircolTM collagen assay were respectively used to detect the amount of fibronectin (FN), connective tissue growth factor (CTGF), and total collagen in each group. Data were processed with one-way analysis of variance, LSD-t test and SNK-q test.Results The optimum MOIs of both lentivirus vectors to HFF-1were50. Comparing with blank control group, TGF-β1up-regulated the highest expressing levels of fibronectin, CTGF and total collagens in positive control group (P<0.05) and negative virus group (P<0.05). This effect is lower in sTβRII group (P<0.05) and Smad4△M4group (P<0.05) and lowest in co-infection group (P<0.05). There was no significant difference between positive control group and negative virus group (P>0.05).Conclusions In HFF-1, both sTβRII and Smad4△M4can reduce the expression of scar related proteins which were up-regulated by TGF-β1. And co-infection of these two genes has a stronger inhibition effect than does single infection, which could be a new strategy for gene therapy of scars.