Experimental Study On Suppression Of TGFβ₁ And TIMP-1 In Hypertrophic Scar By Antisense Therapy

Objective: To suppress the gene expressions of TIMP-1 and TGF β1, promote digression of collagen, and explore the treatment effect on hypertrophic scar of blocking both TIMP-1 and TGF β1.Methods: 1 Anti-TIMP-1 ribozyme genes named Rz182, Rz358, Rz412 and corresponding mutant ribozyme genes were designed and cloned into pBSKneorU6. 32P-labeled TIMP-1 transcripts as targeted RNAs and 32P-labeled ribozyme transcripts were transcribed in vitro, incubated together at different conditions for cleavage reactions. 2 Human hypertrophic scar derived fibroblasts (HF) were stable transfected with reconstructed plasmids named pU6-Rz182, pU6-Rz358 and pU1Rz803. Y irradiation was used to promote transfection efficiency. The transfected cells were examined by MTT and cell cycle to evaluate cell growth. The expressions of mRNA and protein of TIMP-1,TGFβ1 and collagen type I and III were detected using semi-quantity RT-PCR and Western blot.3 Antisense oligodeoxynucletides (ODNs) were designed to inhibit the expression of TIMP-1 and TGF β1, transiently transfected HF. The effect of ODNs to HF was evaluated.Results: 1 Both U6Rzl82 ( Km=29.7 nmol/L, kcat=0.32min-1)and U6Rz358 (Km=39.6 nmol/L, Kcat=0.21 min-1) cleaved the targeted mRNA successfully at 37℃ , while U6Rz412 and mutant ribozymes failed to cleave the targeted mRNA. The cleavage efficiencies (CE) of U6Rzl82 and U6Rz358 were up to 49.23% and 55.21% at 37 ℃. The designed ribozymes possessed perfect specific cleavage activity anti-TIMP-1 in vitro. 2 The ribozymes ere cloned into pEGFPC1 and the vectors were introduced into HF and selected by G418. The anti-TGF β1 ribozyme pU1Rz803 were introduced into HF and selected by Zeocin. In the ribozyme expressing cells, RT-PCR and western blot showed that the TIMP-1 or TGFβ1 expression were significantly depressed, Western blot analysis showed less collagen I/III in the cytoplasm. The growth of the Rz expressing fibroblasts was suppressed. 3 The ODNs successfully inhibited expression of TIMP-1 or TGF β1.Conclusion: 1 The ribozymes prepared in vitro had perfect cleavage activity.2 the ribozymes and ODNs could inhibit the function of TIMP-1 and TGFβ1 significantly. 3 The growth of HF was inhibited significantly after the inhibitions of TIMP-1 or TGF β1.4 After TIMP-1 was suppressed, the degressions of collagen I and III were promoted while the production of collagen was not changed 5 After TGFβ1 was suppressed, collagen degressions were promoted and collagen productions were inhibited. 6 The ribozymes and ODNs prepared in this study have potential application as new therapeutic agents against hypertrophic scar.