The Effect Of P27 On Hypertrophic Scar Fibroblasts

Objective: Scar formation is the result of wound healing, but hypertrophic scar and scar over-contracture often destroy patients' cosmetics or result in patients' dysfunction of organs. Up to till now, there is no effective method to treat scar. With the development in molecular techniques, gene therapy is reported as a hopeful way to treat many diseases. In theory, gene therapy may prevent many kinds of diseases from emerging or developing. Gene therapy is the focus of study in treating tumors and other diseases. The key point for gene therapy is how to select target gene and target cell. p27is a member of the universal cyclin-dependent kinase inhibitor family, and is a putative tumor suppression gene, more and more studies showed that p27 might be a important candidate for gene therapy, but its' effect on scar forming is not clear.we detected p27 protein expression difference between hypertrophic scar and normal skin tissue, we found that p27 was lower in hypertrophic scar tissue, it meanet that p27 might play a role in scar forming, so we selected p27 astarget gene and fibroblast from human hypertrophic scar(HTsFb) as target cell, we constructed the eukaryotic expression plasmid pcDNA3-p27 for tumor suppression gene and transfected p27 into HTsFb to observe the biologic effect of p27 on HTsFb, to provide theoretical support to treat hypertrophic scar with p27 clinically.METHODS: 1.p27 expression was detected in hypertrophic scar and normal skin tissue;2 Synthesized completed p27 cDNA by PCR, and cloned it into pUC19 for DNA sequence analysis, pUC19-p27 and pcDNAS were digested with Hind III and EcoR I, then the p27 cDNA was lignated with pcDNAS at Hind III and EcoR I restriction sites by T4 lignase, and the eukaryotic expression plasmid pcDNAS-p27 for tumor suppression gene p27 formed;3. pcDNA3-p27 for tumor suppression gene p27 was transfected into cultured human hypertrophic scar fibroblasts in vitro, proved its expression of p27 protein by Western Blot and immunohistochemistry;4. (1)MTT and 3H~TdR incorporation were applied to measure the effect of p27 on proliferation and DNA synthesis of HTsFb; (DFlowcytometry (FCM) was applied to analyze the effect of p27 on the cell cycle of HTsFb;(3) We detected the effect of p27 on apoptosis of HTsFb byTerminal deoxynucleotibyl transferase (TdT)-mediated dUTP nickend labeling (TUNED ;(4)In situ hybridization was used to detected the effect of p27on collagen synthesis of HTsFb;(5) Fibroblast-populated collagen lattice(FPLC) model was usedto measure the effect of p27 on contraction of HTsFb;(5)Transmission eletron microscope was applied to observe theeffect of p27 on the ultrastructure of HTsFb.RESULTS: I.p27 protein was lower in hypertrophic scar tissue than normal skin, it meanet that p27 might play a role in scar forming;2. The eukaryotic expression plasmid of p27, pcDNA3-p27, has been constructed successfully;3. pcDNA3-p27 ha been transfected into HTsFb successfully, the stable expression of p27 protein was observed;4.(Dp27 could markedly inhibit proliferation and DNA synthesis of HTsFb;(2)p27 could reduce cellular percentage of S stage, increase cellular percentage of G, and G0 stage. (3)p27 could induce apoptosis of HTsFb (4)p27 could inhibit collagen synthesis of HTsFb; (5)p27 could increase the contraction of fibroblast-populate collagen lattice at early stage, but at late stage, p27 couldinhibit the contraction of fibroblast-populate collagen lattice;(6) p27 could induce the inhibitory characteristic of HTsFb growth.CONCLUSION: By constructing pcDNA3-p27 successfully, we transfected p27 into HTsFb and the transfected HTsFbs could express p27 stablely; with a series of tests such as MTT, 3H-TdR incorporation, FCM, TUNEL, and fibroblast-populated collagen lattice(FPLC) model, we found that p27 might inhibit scar forming by inhibiting proliferation and DNA synthesis of HTsFb, inducing apoptosis of HTsFb, reducing collagen synthesis of HTsFb, inhibiting the contraction of HTsFb. It...