Effects Of Adenovirus-Mediated ShRNA Against HTGF-β₁ Gene Expression On Keloid Fibroblast

Pathological scar (keloid and hypertrophic scar) will form when extracellular matrix (ECM) abnormally deposits after skin destruction. It can demolish our body surface perfection, even bring us function disturbance, and it is always important clinic topic. The cicatrix fibroblast is the key effector cell in the scarring. The dysfunctional fibroblast is known as the reason of abnormal scar formation recently, and transforming growth factorβ(TGFβ) is the most intimate factor in the over-scarring. TGF-β1 can increase ECM synthesis including collagen/fibronectin and hyaluronic acid in vitro experiment. Synthetical TGF-β1 can adjust precollagenⅠ/Ⅲgene express in the scar tissue. Because the relationship between TGF-β1 and scarring exist causal relation, the treatment against TGF-β1 maybe the effective method to cure cicatrix.At present, development of study on gene therapy of abnormal scar has been improved. Many scholars try inhibit TGF-β1 by antisense oligonucleotides and ribozyme, which have low transduction efficiency and transient time. RNA interference (RNAi) technique emergence provides a new way to inhibit gene expression. RNAi is the post transcription gene silence induced by dsRNA, which can silence gene specifically and result in gene functional incapacitation or reduction. RNAi has specificity, persistence and sensitivity. However there are less numerous reports about cure keloid by RNAi in the world. The purpose of this experiment is to inhibit keloid fibroblast's hyperplasia and induce apoptosis by adenovirus-mediated shRNA against hTGF-β1 gene express.This study includes two parts.PartⅠConstruction and preparation of recombinent adenovirus vector of human TGF-β1 shRNAAccording to RNA interference principle, the target sequence was designed, the promoter with shTGFβ1 was synthesized, and human U6 promoter with the fragment including TGF-β1 reverse complement target sequence was obtained by PCR. Then PCR product was linked with the pGEM T Easy vector, which was introduced into competent cell. Sequencing results show that pTU6shTGFβ1(1) was wrong. pTU6shTGFβ1(2) was not measured through completely and it was presumed right. pTU6shTGFβ1(3)and pTU6shTGFβ1(4) were right. AdMax? Adenovirus Creation System was used to construct the replication recombinent adenovirus vector of human TGF-β1 shRNA(rAdTGF-β1). rAdTGF-β1(2),rAdTGF-β1(3),rAdTGF-β1(4) and rAdeGFP titer was 10 9.0 TCID50 /ml,10 8.4 TCID50 /ml,10 9.2 TCID50 /ml,10 8.14TCID50/ml respectively.PartⅡExperiment of rAdTGF transfection keloid fibroblasts in vitroPrimary keloid fibroblasts (target cell) was cultured in vitro. After transfecting with rAdTGF-β1, keloid fibroblasts (KFB) proliferation activity was assayed by MTT. The cells vigor and the apoptosis were detected by flow cytometry. TGF-β1 mRNA level was semiquantitatively assayed by RT-PCR. TGF-β1 protein level was semiquantitatively assayed by Western Blot. Basic control was bland cells. Transfection efficiency was monitored by GFP. Non-specificity inhibition control (rAdTGF-β1(4) was unrelated short hairpin RNA structure.β-actin is inner reference standard. The results show us that, by MTT ,compared with rAdTGF-β1(4), proliferation inhibition rate of KFB transfected by rAdTGF-β1(2) in 24h, 48h, 72h are 15.37%,41.99%,48.43% respectively; and rAdTGF-β1(3) are 8.14%,11.10% and 19.28% respectively; by flow cytometry, compared with rAdTGF-β1(4), G1 period increases obviously, but S period ratio decreases notablely. At the same time, the apoptosis is detected significantly; by RT-PCR, compared with rAdTGF-β1(4), TGF-β1 mRNA relative level of KFB transfected by rAdTGF-β1(2) or rAdTGF-β1(3) in 48h is decreased 94.4% or 57% (p<0.01); by Western blot, compared with rAdTGF-β1(4), TGF-β1 protein relative level of KFB transfected by rAdTGF-β1(2) or rAdTGF-β1(3) in 48h is decreased 74.5% or 43.1%. According to above results, we find that pTU6shTGFβ1(2) is correct though it was not measured completely; rAdTGF-β1(2) inhibiting TGF-β1 presented higher silencing efficiency than rAdTGF-β1(3).On the whole, it can be concluded that adenovirus-mediated shRNA against hTGF-β1 can inhibit proliferation activity of keloid fibroblasts and TGF-β1 protein expression. Moreover, the siRNAs targeting secondary structure region present higher silencing efficiency than those targeting none-secondary structure region.