in the burned area; 3. there was no concomitant disease or history of use of immunosuppressants; 4. before surgery. All the patients were informed of the purpose and procedure of this study and agreed to offer their tissue specimens. The written consent was obtained from all participants involved in this study. All the protocols were approved by the Ethic Committee of Southwest Hospital, Chongqing, China. Preparation of Scar Specimens All the hypertrophic scar specimens and the normal skin specimens were obtained from the same patients who underwent orthopedic surgery at the Institute of Burn Research,and the Department of Plastic and Reconstructive Surgery of Southwest Hospital. The specimens were either washed with PBS and subjected to immediate cell isolation or fixed with saturated trinitrophenol solution and storage in liquid nitrogen. Primary cell culture As previously described [12], fibroblasts were from normal skins (Sk) and hypertrophic scars (Sc). Skin or hypertrophic scar tissue was cut into 0.5 cm3 pieces using a pair of scissors, and the epidermis and dermis were isolated by digestion with 0.25 g/l Dispase II (Sigma, USA). The dermal tissue was minced and digested thoroughly with 30 volumes of 200 U/ml collagenase I solution (Sigma, USA) (200 U collagenase I solution in 1 ml PBS) at 37uC for 2 hours, followed by cell collection by centrifugation. The cells were then cultured in DMEM medium containing 10% calf serum (Hyclone, USA) at 37uC in air containing 5% CO2. The cells were passaged 4 to 9 times and used in the following experiments. Epidermal cells (Ep) were from normal skins. Skin tissue was cut into 1 cm3 pieces using scissors and digested with 2.5 g/l Dispase II (Sigma, USA) at 4uC overnight. The epidermal tissue was collected to be digested by 2.5 g/l trypsin (Sigma, USA) containing 0.2 g/l EDTA?Na2 (Sigma, USA) for 5 minutes. And the epidermal cells were then cultured in K-SFM medium (Gibco, USA) at 37uC in air containing 5% CO2. Peripheral blood monocytes (PBM) were from the healthy peripheral blood. The 10 ml peripheral blood was diluted by 10 ml PBS, followed by adding into 20 ml Fercoll (1.073 g/ml) (TBD, China) gently. The cell suspension was then centrifuged at 900 g for 20 minutes at 20uC. The cells in up-interface were harvested to be diluted by 20 ml PBS. We got the peripheral blood monocytes precipitation after centrifuge the suspension at 700 g for 10 minutes at 20uC. Bone marrow mesenchymal stem cells (mMSC) were from healthy bone marrow and isolated with the same protocol as that for peripheral blood monocytes. The bone marrow mesenchymal stem cells were identified by FACS on the cell markers of CD105 (using FITC-labeled anti-CD105 antibody, 1:50) and of CD34 (using PE-labeled anti-CD34 antibody, 1:10) (Pierce, USA). The corresponding controls were either FITC-IgG2b (1:1000) or PE- IgG1 (1:1000). Preparation of replication defective adenovirus expression vector containing human P311 gene (P311) The P311 plasmid was a kind gift from Professor Matthieu Levi- Srauss (Inserm U114, Chaire de Neuropharmacologie, College de France, 11, Place Marcelin Berthelot, 75005 Paris, France). pAdTrack-CMV plasmid and pAdEasyTMxL Adenoviral Vector System were purchased from Stratagene (USA). The P311 plasmid was digested and ligated with T vector. After the recombinant was identified by sequencing, pAdTrack-CMV/pGEM T-Easy-P311 shuttle plasmid was constructed and transfected into competent E. coli DH5a (ATCC, USA), followed by collection of rigid clones. The Role of Human P311 Gene Then, pAdTrack-CMV/pGEM T-Easy-P311 shuttle plasmid and adenovirus genome-containing pAdEasy-P311 plasmid underwent homologous recombination in E. coli BJ5183, and adenovirus plasmid expressing the target gene was screened. The virus was packaged and amplified in AD-293 cells, and the virus titer was determined by means of Plaque Forming Unit(PFU). Transduction of fibroblasts with replication defective adenovirus expression vector Fibroblasts were digested with 0.25 g/l trypsin, and inoculated into 75 cm2 culture flasks at a density of 16106 cells in 10 ml DMEM medium containing 10% calf serum. Upon 80% cell confluence, the medium was discarded, and the cells were washed with 0.1 M PBS and antibiotic-free, serum-free DMEM medium. Then, 16109 PFU/ml virus stock suspension was added to cover the cells on the culture flask bottom. The cells were cultured for 1 hour at 37uC in air containing 5% CO2, and then the virus suspension was removed, followed by the addition of DMEM medium containing 10% calf serum, and cultured in an incubator. At the indicated time points, the cultures were microscopically examined for the expression of green fluorescent protein using an inverted microscope (Olympus, Japan). Preparation of the model of fibroblast populated collagen lattice (FPCL) and measurement of fibroblast contraction in FPCL Model The FPCL model was used to observe the contractile ability of fibroblasts. As described previously [13], soluble collagen was extracted from the tails of SD rats. The cell suspension (the final concentration: 56105 cells/ml), calf serum (the final concentra- tion: 10%), 56DMEM, and collagen protein solution were mixed at a volume ratio of 1:1:2:6 at 4uC and the pH of the mixture was adjusted to 7.2. The fibroblast/collagen protein suspension was added into culture dishes at a size of 35 mm in diameter, and maintained for 10 minutes at 37uC in air containing 5% CO2 until gel formation, followed by addition of 1 ml DMEM medium containing 10% calf serum and further cultured at 37uC in air containing 5% CO2. The diameter of FPCL was recorded every other day. The FPCL contraction index is calculated as follows [14]: CI = [12(D/D0)2]6100% (CI: contraction index; D: diam- eter of collagen protein gel block; D0: initial diameter of gel block (35 mm)). Silencing of P311 in fibroblast by shRNA This study used three P311 shRNAs, targeting different regions of the P311 transcript and one control shRNA contracted shRNA1, shRNA 2, shRNA 3 and control. The sequences of shRNA1 were 59-GATCCGCCGCAAGAAGAACGATGAGT- TCAAGACGCTCATCGTTCTTCTTGCGGTTTTTTGTCG- ACA-39 and 39-GCGGCGTTCTTCTTGCTACTCAAGTTC- TGCGAGTAGCAAGAAGAACGCCAAAAAACAGCTGTTC- GA-59. shRNA2 sequences were 59-GATCCGAACGATGAGA- CAAACGCTTTCAAGACGAGCGTTTGTCTCATCGTTCT- TTTTTGTCGACA-39 and 39-GCTTGCTACTCTGTTTGC- GAAAGTTCTGCTCGCAAACAGAGTAGCAAGAAAAAACA- GCTGTTCGA-59. shRNA3 sequences were 59-GATCCGAA- GAACGATGAGACAAACTTCAAGACGGTTTGTCTCATC- GTTCTTCTTTTTTGTCGACA-39 and 39-GCTTCTTGCT- ACTCTGTTTGAAGTTCTGCCAAACAGAGTAGCAAGAA- GAAAAAACAGCTGTTCGA-59. And the sequences of the control shRNA were 59-GATCCGACTTCATAAGGCGCATG- CTTCAAGACGGCATGCGCCTTATGAAGTCTTTTTTGT- CGACA-39 and 39-GCTGAAGTATTCCGCGTACGAAGTT- PLoS ONE | www.plosone.org 2 April 2010 | Volume 5 | Issue 4 | e9995