Study on the Role of P311 in the Formation of Hypertrophic Scar
|School||Third Military Medical University|
|Keywords||hypertrophic scar fibroblast P311 gene adenovirus gene transfection RNA interference Fibroblast-populated collagen lattice|
The formation of hypertrophic scar is an abnormal wound-healing response after thermal injuries or partial-thickness wounds. In predisposed individuals, however, these abnormal wound-healing processes show a kind of over-healing and produce an overabundant wound matrix responsible for raised, red, and inflexible scar tissue, which causes itching and pain, and might lead to serous functional and cosmetic problems for the patient. For reason of unclear pathogenesis, it still could not be controlled with a satisfying result. Studies have implicated that multiple cells involve in the wound-healing process, such as fibroblasts, macrophages, endotheliocytes, mast cells, lymphocytes, etc. Abnormal activity of these cells may lead to the formation of hypertrophic scar. It’s interesting that hypertrophic scar exclusively develop in humans with a feature of joint contracture. Under microscope, hypertrophic scar exhibit nodular structures in which fibroblastic cells, small vessels, and fine, randomly organized collagen fibers are present. Dr. Shin isolates and concentrates the fibroblasts that express α-smooth muscle actin from the hypertrophic scar derived fibroblasts by using the magnetic activating cell-sorting column to obtain the myofibroblast group. When the cells are cultured in the fibroblast-populated collagen gel, the myofibroblast group, compared with the fibroblast group, showed statistically significant contraction of the collagen gel lattice day by day. It is widely acknowleged that fibroblasts have an essential role in wound contraction, but the exact involvement of fibroblasts in this contraction has not been adequately elucidated. We compared differentially expressed genes between normal skin and hypertrophic scar from 5 patients with microarry containing 4096 human genes. Ninety-seven differentially expressed genes were found in our study, involving proto-oncogene genes, metabolism related genes, cytoskeletal genes, immune related genes, et al. A unknown gene, P311, is out-standing among all these hypertrophic scar related genes. P311 is highly over-expressed in the early hypertrophic scar. The Cy5/Cy3 ratio of P311 on the microarry reaches 9.1338, meanwhile, the average ratio is 2.000. It implies that P311 may play an important role on hypertrophic scar formation. P311 does not belong to any known family of proteins; therefore, it does not provide clues suggesting function. PEST domain on the N-terminus of P311 gene is highly conserved across human, mouse, and chicken species. The domain is targeted by the ubiquitin/proteasome pathway(UPP) and is mainly found in short-lived proteins such as transcription factors, cytokines, and signaling molecules. It is known that transforming growth factor beta (TGF-β) signaling plays a fundamental role in hypertrophy scar formation. Smads pathway is the main signal pathway of TGF-βto transfer signals from cell membrane of fibroblasts to the nucleus. After the Smad proteins are expressed in cell, they are rapidly degraded by UPP in order to maintain the rhythms of regular signal transduction. Therefore, P311 may involve in hypertrophic scar formation by interfering the signal pathway of TGF-β.Dr. Pan reported that P311 gene could induce myofibroblats transformation from fibroblasts. Recently, Dr. Fujitani found that P311 gene could accelerate nerve regeneration of the axotomized facial nerve. These findings can explain some clinical problem of hypertrophic scar, such as: proliferation of myofibroblasts, itching and pain from regeneration of nerve on wounds. All these evidences imply that P311 gene may play a role on the formation of hypertrophic scar. But what is exactly role of P311 gene on hypertrophic scar formation? We’ll study on the role of P311 gene involving hypertrophic scar by answering the following questions: ①Which cells in the hypertrophic scar express P311 gene? ②Which kinds of changes will occur, when the P311 gene is up-regulated in the target cells? ③What will happen, when the P311 gene is down-regulated in the target cells? Methods: 1. Which cells in the hypertrophic scar express P311 gene? ①To detect P311 mRNA in the hypertrophic scar by in situ hybridization. ②To detect P311 protein in the hypertrophic scar by immunohistochemical staining. ③To detect P311 mRNA among cells in skin from different embrionary origin by RT-PCR. These cells are human skin derived fibroblasts, hypertrophic scar derived fibroblasts, epidermal cells, peripheral mononuclear cells, mesenchymal stem cells. 2. Which kinds of changes will occur, when the P311 gene is up-regulated in thetarget cells? ①Construction and identification of recombinant adenovirus which contains human P311 genes. ②Induction of P311 gene to human skin derived fibroblasts and hypertrophic scar derived fibroblasts by the recombinant adenovirus. ③Measure the contraction of FPCL which contains fibroblast with up-regulated P311 gene. ④Detection of α-SMA、TGF-β1、α1(Ⅰ)collagen gene(COL1A1) mRNA on fibroblast with up-regulated P311 gene by RT-PCR. Cell cycles of these cells were also detected by flow cytometry. 3. What will happen, when the P311 gene is down-regulated in the target cells? ①Construction of siRNA plasmid vectors HK, MB1, MB2, MB3 targeted on P311 gene. ②Transfection of siRNA plasmid vector into hypertrophic scar derived fibroblasts by liposome transfection technique. The transfected cells were continuously cultured under G418 for 3 weeks. The cells stably expressed siRNA plasmid vector genes were selected by EGFP expression. P311 mRNAs and proteins in these cells were detected by RT-PCR and western blot. The cells in which P311 gene were inhibited were the target cells in the following study. ③Measure the contraction of FPCLs which contains fibroblasts with down-regulated P311 gene. ④Detection of TGF-β1 mRNA on fibroblasts with down-regulated P311 gene by RT-PCR. Results: 1. Which cells in the hypertrophic scar express P311 gene? It is fibroblast cells which express P311 gene in the hypertrophic scar. The early hypertrophic scar contains a high density of these cells. The number of fibroblasts expressed P311 gene decreased with time. Only the human hypertrophic scar derived fibroblasts express P311 mRNA, not the human skin derived fibroblasts, epidermal cells, peripheral mononuclear cells, or mesenchymal stem cells. 2. Which kinds of changes will occur, when the P311 gene is up-regulated in thetarget cells? We successfully constructed recombinant adenovirus which could induce human P311 protein expression in 293 cells. The skin derived fibroblast cells were transfected by recombinant adenovirus which contained human P311 gene and then were put into FPCL system. It was observed that the gel had contracted significantly after P311 genes were up-regulated in these cells. It was also found that the expression of α-SMA、TGF-β1 and α1(Ⅰ)collagen gene(COL1A1) mRNA increased after the fibroblasts were transfected by adenovirus. Besides, the percentage of the cells in S stage was also increased. Under microscope, the transfected cell viewed as an activated cell. 3. What will happen, when the P311 gene is down-regulated in the target cells? We successfully constructed siRNA plasmid vectors HK, MB1, MB2, MB3 targeted on P311 gene. After the siRNA plasmid vectors had been transfected into hypertrophic scar derived fibroblasts by liposome transfection technique, the cells in which P311 gene were inhibited satisfiedly were selected. It was observed that contraction of FPCL system had decreased significantly after the cells with down-regulated P311 gene expression had been seeded into the gel. It was also found that the expression of TGF-β1 mRNA decreased in these cells. Conclusion: 1. It is fibroblast cells which express P311 gene in the hypertrophic scar. 2. We successfully constructed recombinant adenovirus which could induce human P311 protein expression in 293 cells. 3. We successfully constructed siRNA plasmid vectors targeted on P311 gene. 4. P311 gene can induce transformation of myofibroblast from fibroblast. 5. Our hypothesis: P311 gene inhibits degradation of Smad proteins in hypertrophic scar derived fibroblast cells through UPP pathway. It is well known that Smad pathway is the predominant pathway of TGF-βsignal transduction. As a result, TGF-βsignal is abnormally amplified in the fibroblasts. Reasonably, the cells in hypertrophic scar can transform from fibroblasts to myofibroblast with enhanced expression of α-SMA, TGF-β, COL1A1 mRNA and contracture power. Besides, P311 may play a role in the formation of hypertrophic scar by accelerating TGF-βautoinduction. Because of the two reasons, P311 exacerbates occurrence of hypertrophic scar. However, the derailment of the protectingwound-healing processes simulates fibroblasts to produce P311.